23 Jul 2019: Zeidan Q, He F, Zhang F, Zhang H, Jacobson A, et al. (2019) Correction: Conserved mRNA-granule component Scd6 targets Dhh1 to repress translation initiation and activates Dcp2-mediated mRNA decay in vivo. PLOS Genetics 15(7): e1008299. https://doi.org/10.1371/journal.pgen.1008299 View correction
Scd6 protein family members are evolutionarily conserved components of translationally silent mRNA granules. Yeast Scd6 interacts with Dcp2 and Dhh1, respectively a subunit and a regulator of the mRNA decapping enzyme, and also associates with translation initiation factor eIF4G to inhibit translation in cell extracts. However, the role of Scd6 in mRNA turnover and translational repression in vivo is unclear. We demonstrate that tethering Scd6 to a GFP reporter mRNA reduces mRNA abundance via Dcp2 and suppresses reporter mRNA translation via Dhh1. Thus, in a dcp2Δ mutant, tethered Scd6 reduces GFP protein expression with little effect on mRNA abundance, whereas tethered Scd6 has no impact on GFP protein or mRNA expression in a dcp2Δ dhh1Δ double mutant. The conserved LSm domain of Scd6 is required for translational repression and mRNA turnover by tethered Scd6. Both functions are enhanced in a ccr4Δ mutant, suggesting that the deadenylase function of Ccr4-Not complex interferes with a more efficient repression pathway enlisted by Scd6. Ribosome profiling and RNA-Seq analysis of scd6Δ and dhh1Δ mutants suggests that Scd6 cooperates with Dhh1 in translational repression and turnover of particular native mRNAs, with both processes dependent on Dcp2. Our results suggest that Scd6 can (i) recruit Dhh1 to confer translational repression and (ii) activate mRNA decapping by Dcp2 with attendant degradation of specific mRNAs in vivo, in a manner dependent on the Scd6 LSm domain and modulated by Ccr4.
Previous work showed that Scd6 homologs in Drosophila, Xenopus, and humans are associated with translationally repressed mRNAs in RNA granules, and that they interact with other mRNA silencing factors, including homologs of RNA helicase Dhh1. However, there is little evidence that such Scd6 homologs are critical for translational repression or degradation of specific mRNAs in vivo. Yeast Scd6 interacts with the mRNA decapping enzyme and was shown to inhibit translation in cell extracts through binding to cap-binding translation initiation factor eIF4G. However, it was unknown whether Scd6 represses translation or accelerates degradation of any specific mRNAs in vivo, or whether Scd6 requires the decapping enzyme or its activators for such events. Here we show that tethering Scd6 stimulates the degradation or translational repression of reporter mRNAs in yeast, collaborating with Dhh1 for translational repression and the decapping enzyme Dcp2/Dcp1 for mRNA turnover. Using ribosome profiling we further identify groups of native mRNAs that appear to be targeted for degradation or translational repression by Scd6 and Dhh1 and find unexpectedly that Dcp2 is necessary for translational repression as well as enhanced degradation of most such mRNAs. Thus, Scd6 partners with Dhh1 and Dcp2 to mediate translational repression and degradation of specific mRNAs in vivo.
Citation: Zeidan Q, He F, Zhang F, Zhang H, Jacobson A, Hinnebusch AG (2018) Conserved mRNA-granule component Scd6 targets Dhh1 to repress translation initiation and activates Dcp2-mediated mRNA decay in vivo. PLoS Genet 14(12): e1007806. https://doi.org/10.1371/journal.pgen.1007806
Editor: Kristian Baker, Case Western Reserve University, UNITED STATES
Received: June 1, 2018; Accepted: November 2, 2018; Published: December 7, 2018
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: RNA-Seq and ribosome profiling data can be obtained from the GEO database under accession numbers GEO:GSE107841, GEO:GSE114428, and GEO:GSE114892. All other relevant data are within the paper and its Supporting Information files.
Funding: This work was supported in part by the Intramural Research Program of the National Institutes of Health (AGH) and by National Institute of General Medical Sciences Grants GM27757-37 and GM122468-01 (AJ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
After being transcribed and processed in the nucleus, and exported to the cytoplasm, mRNAs can either engage with the translational machinery for protein synthesis, undergo storage in a translationally silent state, or be targeted for degradation. Cellular mRNAs can alternate between these processes, and translation, storage, and decay influence each other in multiple ways to regulate gene expression . In general, mRNAs selected for translation are thought to establish interactions of their 5’-cap and 3’-poly(A) tail appendages with proteins from the translational machinery. mRNAs are activated for translation by binding to the mRNA cap of eukaryotic initiation factor eIF4F (comprised of cap-binding protein eIF4E, scaffolding protein eIF4G, and helicase eIF4A) and association of poly(A)-binding protein (PABP) with the poly(A) tail; and interactions between eIF4G and PABP can form a “closed-loop” mRNP competent for initiation. Further interactions between eIF4G and other translation initiation factors associated with the 43S pre-initiation complex (PIC) pre-assembled on the small (40S) ribosomal subunit serve to recruit mRNA and form a 48S PIC competent for subsequent mRNA scanning and start codon selection (reviewed in ).
The mRNA decay machinery can compete with the translation initiation machinery for access to cap and poly(A) tail of the transcript . Degradation of mRNA is generally initiated by removal of the poly(A) tail through sequential deadenylation reactions by the Pan2/Pan3 and Ccr4-Not complexes, followed by loss of associated PABP . Turnover can proceed in the 3’ to 5’ direction via the cytoplasmic exosome, or in the 5’ to 3’ direction via removal of the cap by the Dcp2/Dcp1 enzyme complex and exonucleolytic digestion by Xrn1 . Decapping is a highly regulated step that irreversibly commits mRNAs for complete digestion . One regulatory mechanism is thought to include formation of complexes comprised of Dcp1/2, Xrn1, and distinct sets of Dcp2 interactors that modulate both mRNA substrate recruitment or catalysis by Dcp2 [4, 6]. These decapping activators include DEAD-box helicase Dhh1, Pat1, Edc3, the Lsm1-7 complex, and Scd6 . Decapping activators can function by distinct mechanisms: (i) Scd6, Dhh1, and Pat1 can inhibit translation initiation by blocking formation of a 48S PIC in vitro, which in turn favors mRNA decapping ; (ii) Pat1 and Edc3 can directly bind Dcp2 and stimulate its catalytic activity in vitro and in vivo [7–9]; and (iii) Dhh1 can detect reductions in ribosome transit at non-optimal codons and further inhibit translation elongation, eliciting acceleration of transcript decapping in vivo [6, 10].
Based on their ability to inhibit translation initiation in vitro, it has been proposed that the decapping activators Dhh1 and Scd6 can direct their target mRNAs from a translationally active state to an mRNP state competent for mRNA storage or decapping . Scd6 belongs to a highly conserved protein family with orthologs in humans (hRAP55/Lsm14), Xenopus laevis (xRAP55), Drosophila melanogaster (Tral), Caenorhabditis elegans (CAR-1), Trypanosoma brucei (TbSCD6), and fission yeast (Sum2), among others . All family members contain a conserved N-terminal LSm (like-Sm) domain, followed by central DFDF-FFD-TGF boxes, and variable numbers of C-terminal RGG/RGX motifs [12, 13]. In metazoans and Plasmodium, Scd6 associates with Dhh1 homologs, other proteins, and mRNAs to form stable mRNPs that contain translationally silent transcripts subject to developmental regulation . For example, in Drosophila, Scd6 and Dhh1 homologs, Tral and Me31B, belong to a repression complex containing CUP, an inhibitor of eIF4E-eIF4G association, shown to control the translation of certain mRNAs with key functions in embryogenesis [14, 15]. This complex might function more broadly to repress translation of many mRNAs during early embryogenesis, possibly by coating the mRNA [16, 17]. An Scd6 homolog in Xenopus, xRAPB, associates with the Dhh1 ortholog Xp54 in translationally inactive maternal transcripts in stored mRNPs; although xRAPB appears to oppose rather than promote Xp54 function in repressing translation in oocytes . A longer Xenopus variant, xRAP55 represses translation in vitro, and decreases reporter protein levels when tethered to a reporter mRNA in cell extracts, dependent on its N-terminal LSm domain ; however, its role in oocytes is unclear .
In yeast and humans, Scd6 is required for the formation and accumulation of mRNA-containing cytoplasmic aggregates called Processing (P) bodies, and it also localizes to mRNA-containing stress granules under a variety of adverse conditions [19–22]. In addition to its ability to bind the decapping enzyme subunit Dcp2, yeast Scd6 can also interact with the other decapping activators Pat1 and Edc3 [7, 23–29], as well as with various members of the Lsm complex and Dhh1 [23, 24, 29]. In vitro, yeast Scd6 represses translation initiation by directly interacting with the C-terminal region of eIF4G (in the context of eIF4F) via the Scd6 C-terminal RGG domain, preventing recruitment of the 43S PIC to activated mRNA and formation of the 48S PIC [7, 22]. The RGG domain is also required for overexpressed Scd6 to inhibit cell growth and produce stress granules . Yeast Scd6 can also interact with other translation components, including proteins of the small and large ribosomal subunits, PABP, eIF4B, eIF5, and eEF1A [23, 25, 29]. Like yeast Scd6, the Arabidopsis homolog, Dcp5, was shown to repress translation in vitro . The homolog in Trypanosoma brucei is present in cytoplasmic granules and appears to be a general repressor of translation, even though it does not exhibit an association with the Dhh1 homolog [13, 16, 17] that is otherwise conserved in yeast, worms, flies, and vertebrates .
Considerable evidence points to an important role for yeast Scd6 in cytoplasmic post-transcriptional control. It interacts with the decapping enzyme and its regulators, can inhibit 48S PIC assembly in vitro, and overexpression of its gene induces stress granule formation and inhibits cell growth in a manner requiring its C-terminal eIF4G-interaction domain. Nevertheless, it has not been demonstrated that Scd6 mediates the degradation or translational repression of any specific mRNA in yeast cells. In this study, we show that tethering Scd6 to two different reporter mRNAs in vivo evokes Dhh1-dependent repression of translation initiation, which is accompanied by reporter-specific Dcp2-mediated mRNA turnover. Both Scd6 functions are dependent on its LSm domain, and are enhanced by depletion of the Ccr4 deadenylase of the Ccr4-Not complex in vivo. By ribosome profiling we further provide evidence that Scd6 cooperates with Dhh1 in repressing the mRNA abundance and translation of particular native mRNAs in nutrient-replete medium, and present evidence that both functions require Dcp2. Thus, Scd6 appears to be an important component of the gene expression control network in yeast cells, acting at the levels of both translation initiation and mRNA turnover.
Tethered Scd6 represses expression of a GFP reporter mRNA
Previously, it was shown that Scd6 interacts with mRNA decapping factors and can inhibit translation initiation in vitro [7, 22]; however, it was unknown whether Scd6 can reduce the abundance and repress the translation of specific mRNAs in vivo. Because native mRNA substrates of Scd6 were unknown, we employed a tethered-function assay to examine the consequences of Scd6 binding to reporter mRNA—an approach used successfully to demonstrate regulation of mRNA translation and turnover by the decapping activator/translational repressor Dhh1 . A fusion of Scd6 to bacteriophage MS2 coat protein (CP), or MS2 CP alone, each tagged with three FLAG epitopes (henceforth, Scd6-MS2-F or MS2-F), was expressed in wild-type (WT) cells from mRNA containing the 5’ and 3’ untranslated regions (UTRs) of SCD6 driven by the native SCD6 promoter from a low copy plasmid. A GFP reporter mRNA, harboring the 5’UTR and 3’UTR of PGK1 mRNA with tandem MS2 RNA recognition elements inserted in the 3’ UTR, and driven by a galactose-inducible GAL1 UAS and PGK1 hybrid promoter , was expressed from a low copy plasmid in the same strains by culturing cells with galactose as carbon source. As a positive control, we expressed the Dhh1-MS2 fusion shown previously to evoke translational repression of the same GFP reporter . Western blotting with anti-GFP antibodies and either Northern blotting or qRT-PCR analyses were conducted to measure steady-state expression of GFP protein and GFP reporter mRNA, respectively, and the ratio of these measurements yielded the translational efficiency (TE) of the reporter mRNA (Fig 1A).
(A) Schema of the MS2 CP tethering assay with Scd6-MS2-F fusion protein targeted to stem-loops in the 3’ UTR of the GFP reporter; with summary of assays used to measure GFP protein or mRNA abundance and translation efficiency (TE) of the reporter. (B) WT cells (BY4741) were co-transformed with an expression plasmid for MS2(2μ) (pJC398), Dhh1-MS2 (pJC236), Scd6-MS2-F (pQZ127) or MS2-F (pQZ130), along with GFP reporter plasmid (pJC429), cultured in synthetic complete medium without leucine or uracil (SC-L-U) with 2% galactose/2% raffinose replacing dextrose at 30°C for at least two cell divisions, and harvested at A600 ~0.6–0.7. Total protein was extracted under denaturing conditions and subjected to Western blot analysis with antibodies against FLAG, GFP or Prt1 (as loading control). Typical results from three biological replicates are shown. (C) Relative GFP protein signal normalized to Prt1 protein signal by densitometry analysis of samples in (B). Average results (± S.E.M.s) from at least three biological replicates are shown. (D) Transformants from (A), and WT strain BY4741 harboring empty vector YCpLac111 and GFP reporter pJC429, were cultured as in (B). Total RNA was extracted from whole cell extracts (WCEs) and reporter mRNA abundance was quantified by RT-qPCR, relative to that of ACT1 mRNA. Average results (± S.E.M.s) from at least three biological replicates are shown. (E) Total RNA samples from (D) were subjected to Northern blot analysis and probed for GFP mRNA; blots were stripped and re-probed for PYK1 mRNA as a loading control. (F) Changes (Δ) in expression of GFP reporter protein or mRNA, or reporter TE, on tethering Scd6-MS2-F, as ratios of values in cells harboring Scd6-MS2-F versus MS2-F. Mean values (± S.E.M.s) were determined from at least three biological replicates. Calculations of S.E.M.s for mean ratios of GFP protein and mRNA expression shown in (F), and determination of P-values from significance testing of differences in mean values in (C-D) using an unpaired Student’s t-test, were conducted as described in the supporting file S1 Text “Analysis and Explanation of Supporting Data Files”. P-values are summarized as: **, P <0.01; *, P <0.05. P-values for these and all other statistical tests performed in this study can be found in the supplementary data files provided for the respective figures.
Tethering Scd6-MS2-F reduced expression of GFP protein and reporter mRNA by comparable amounts, ~2.5- to 3-fold, compared to tethering MS2-F alone (Fig 1B–1D, Scd6-MS2-F vs. MS2-F; P <0.0001 in C & D). These results are similar to those observed on tethering Dhh1-MS2 versus MS2 alone expressed from a high-copy plasmid (Fig 1B–1D, Dhh1-MS2 vs. MS2(2μM)), in agreement with previous findings . Expression of GFP protein and mRNA in cells expressing MS2-F was essentially indistinguishable from that measured in transformants of the same strain harboring empty vector (S1A Fig, Fig 1C and 1D). Northern analysis confirmed the repression of GFP mRNA by both Scd6-MS2-F and Dhh1-MS2 in comparison to empty vector and MS2-alone controls (Fig 1E). These results are consistent with the possibility that tethering Scd6 increases the rate of GFP mRNA turnover to lower its steady-state abundance, with attendant reduction in GFP protein expression. Owing to comparable repression of reporter protein and mRNA, tethered Scd6-MS2-F evokes little change in the TE of the GFP reporter (Fig 1F).
To determine whether tethering Scd6-MS2 to GFP mRNA accelerates its degradation, we measured the half-life of GFP mRNA following a shift from galactose to glucose medium that should repress the GAL promoter and halt new synthesis of reporter mRNA. On tethering Scd6-MS2-F, the half-life of GFP mRNA was significantly reduced from 2.8 ± 0.06 min (tethering MS2-F alone) to 1.9 ± 0.15 min (P = 0.03; S1B Fig). A similar fold-reduction in half-life was observed on tethering Dhh1-MS2 vs MS2 alone (2.3 ± 0.09 min vs. 3.20 ± 0.02 min, respectively; P = 0.01; S1C Fig). We conclude that tethered Scd6-MS2-F reduces GFP mRNA abundance by accelerating its degradation.
Because it was shown previously that Npl3 and Sbp1 can also repress translation initiation in vitro dependent on interaction of their RGG domains with eIF4G in a manner similar to Scd6 , we examined MS2 fusions of these proteins, expressed from plasmid constructs containing the native promoters and 5’- and 3’-UTRs of NPL3 or SBP1, respectively, along with the corresponding MS2 control proteins. In contrast to results obtained for Scd6-MS2-F, neither Npl3-MS2-F nor Sbp1-MS2-F had any significant effect on GFP expression from the same reporter analyzed above (S2A, S2B, S2C and S2D Fig) despite being expressed at levels exceeding that of Scd6-MS2–F (S2E Fig). These results underscored the specificity of the effects of Scd6-MS2-F and led us to probe its mechanism of repression.
Tethered Scd6 evokes Dcp2-dependent reduction of reporter mRNA abundance and Dhh1-dependent translational repression
As Scd6 can bind to Dcp2, the catalytic component of the decapping enzyme , we asked whether the repression of reporter mRNA abundance evoked by tethered Scd6-MS2-F is attenuated in a dcp2Δ strain. The isogenic WT DCP2 strain employed for this experiment exhibited reductions in GFP mRNA and GFP protein expression conferred by Scd6-MS2-F versus MS-F alone (Fig 2A–2D, WT data) equal to, or greater than those observed in the different WT strain employed above (Fig 1B–1D). Tethering Scd6-MS2-F to the GFP reporter in dcp2Δ cells reduced the abundance of GFP mRNA (Fig 2C, dcp2Δ, Scd6-MS2-F vs. MS2-F; P = 0.03). Importantly, however, the reduction in reporter mRNA abundance conferred by Scd6-MS2-F was diminished in dcp2Δ versus WT cells, with the ratio of GFP mRNA in cells expressing Scd6-MS2-F versus MS2-F increasing from ~0.4 in WT to ~0.7 in dcp2Δ cells (Fig 2D, white bars, WT vs. dcp2Δ; P = 0.009). Expression of GFP protein in cells containing MS2-F alone was reduced somewhat in the dcp2Δ vs. WT strain; however, this reduction occurred independently of tethering as it was also observed in the corresponding strains containing empty vector (S3A and S3B Fig). Tethering Scd6-MS2-F in dcp2Δ cells conferred a strong reduction in GFP protein expression compared to MS2-F alone (Fig 2B, dcp2Δ, Scd6-MS2-F vs. MS2-F; P = 0.002), that was only slightly smaller in magnitude compared to that seen in WT cells (Fig 2D, black bars, WT vs. dcp2Δ). As a consequence of relatively greater repression of GFP protein versus GFP mRNA on tethering Scd6-MS2-F versus MS2-F alone in the dcp2Δ mutant (Fig 2D, WT vs. dcp2Δ, black vs. white bars), the TE of GFP reporter mRNA is diminished in dcp2Δ versus WT cells by ~33% (Fig 2E, WT vs. dcp2Δ; P = 0.005). These findings suggest that: (i) Dcp2 is required for strong repression of reporter mRNA abundance, and (ii) translational repression of the GFP mRNA is unveiled in cells lacking Dcp2, where mRNA turnover by tethered Scd6-MS2 is diminished. Similar conclusions were reached previously for tethered Dhh1-MS2 .
(A-C) Transformants of WT (HFY114), dcp2Δ (CFY1016), dhh1Δ (YQZ127), or dcp2Δdhh1Δ (QZY128) strains (isogenic in the W303 background) harboring expression plasmids for MS2-F (pQZ130] or Scd6-MS2-F (pQZ127) and GFP reporter pJC429, were analyzed for reporter protein (A-B) and mRNA (C) expression as in Fig 1B–1D. Mean values (± S.E.M.s) from three different biological replicates per group are shown. (D-E) Changes in expression of GFP reporter protein or mRNA (D), or in TE (E), on tethering Scd6-MS2-F versus MS2-F for the strains analyzed in panels A-C, reported as in Fig 1F. Mean values (± S.E.M.s) were determined from at least three biological replicates. Calculations of S.E.M.s for changes in mean ratios of GFP protein and mRNA expression shown in (D), and determination of P-values from significance testing of differences in mean values in (B-E) using an unpaired Student’s t-test, were conducted as described in the supporting file S1 Text “Analysis and Explanation of Supporting Data Files”. P-values are summarized as: **, P <0.01; *, P <0.05; n.s., not significant. In (E), the entries directly above the bars in cols. 2–3 refer to differences in mean values between the indicated mutants vs. WT (col. 1).
The concordance between effects on GFP reporter mRNA and protein expression conferred by tethered Scd6-SM-2 (Fig 2A–2E) and Dhh1-MS2 (Sweet et al., 2012) in WT and dcp2Δ cells raised the possibility that Dhh1 is required for translational repression conferred by tethered Scd6-MS2-F. To address this possibility, we examined the regulation of GFP reporter expression in dhh1Δ cells. In contrast to our findings for isogenic dcp2Δ cells, we observed greater repression of reporter mRNA abundance by tethered Scd6-MS2-F in dhh1Δ versus WT cells (Fig 2C, Scd6-MS2-F vs. MS2-F, WT vs. dhh1Δ), with the Scd6-MS2-F/MS2-F repression ratio for GFP mRNA declining ~2.4-fold from ~0.4 in WT to ~0.17 in dhh1Δ cells (Fig 2D, white bars, WT vs. dhh1Δ; P = 0.007). Thus, unlike Dcp2, Dhh1 is dispensable for repression of reporter mRNA abundance by tethered Scd6-MS2-F. One possible explanation for the enhanced repression of GFP mRNA in dhh1Δ cells (vs. WT) might be that Dhh1 impedes Dcp1/Dcp2-mediated decapping of the reporter in the presence of tethered Scd6-MS2-F. Repression of GFP protein by Scd6-MS2-F was also intact in the dhh1Δ strain (Fig 2B, Scd6-MS2-F vs. MS2-F; P = 0.001), with an Scd6-MS2-F/MS2-F repression ratio for GFP protein ~1.6-fold lower than that seen in WT cells (Fig 2D, black bars, WT vs. dhh1Δ; P = 0.03). However, owing to even greater Scd6-MS2-F-mediated repression of GFP mRNA compared to GFP protein, the TE of the GFP reporter was increased by ~1.4-fold in dhh1Δ vs. WT cells (Fig 2E; P = 0.009). This observation is consistent with the possibility that Dhh1 contributes to translational repression by tethered Scd6-MS2-F.
We reasoned that if Dcp2 and Dhh1 are respectively required for repression of mRNA abundance and translation by tethered Scd6-MS2-F, then eliminating both proteins in a dcp2Δ dhh1Δ double mutant should abrogate repression of both GFP protein and GFP mRNA by Scd6-MS2-F. Indeed, nearly identical expression levels of GFP protein and mRNA were observed in the dcp2Δ dhh1Δ mutant whether expressing Scd6-MS2-F or MS2–F (Fig 2B and 2C, dcp2Δ dhh1Δ, Scd6-MS2-F vs. MS2-F), yielding near-unity Scd6-MS2-F/MS2-F repression ratios in this strain for both reporter mRNA and protein expression (Fig 2D, dcp2Δ dhh1Δ, black and white bars), which are markedly increased from the corresponding repression ratios of 0.36 and 0.40 in WT cells (Fig 2D, dcp2Δ dhh1Δ vs. WT, white and black bars; P = 0.004 (Protein), P = 0.0009 (mRNA)). Importantly, comparing the reporter TEs in the double mutant to the dcp2Δ single mutant reveals a loss of translational repression in dcp2Δ dhh1Δ cells (Fig 2E, dcp2Δ dhh1Δ vs. dcp2Δ; P = 0.04), supporting a requirement for Dhh1 in translational repression of GFP mRNA abundance by tethered Scd6-MS2-F. That the residual repression of mRNA abundance seen in the dcp2Δ single mutant is eliminated by dhh1Δ in the double mutant (Fig 2D, white bars, dcp2Δ dhh1Δ vs. dcp2Δ; P = 0.02) might indicate that Dhh1 mediates a Dcp2-independent mechanism of mRNA degradation evoked by tethered Scd6-MS2-F in dcp2Δ cells, such as involvement of the exosome or a ribosome-associated endonuclease. In summary, analyses of dcp2Δ and dhh1Δ mutants indicate that efficient repression of reporter mRNA abundance is dependent on Dcp2, whereas Dhh1 participates in translational repression, by tethered Scd6-MS2-F.
Tethered Scd6-MS2-F alters the polysomal distribution of GFP mRNA
Having observed that the occurrence of translational repression of GFP mRNA is unmasked in dcp2Δ cells, owing to partial stabilization of reporter mRNA, we examined the effect of tethered Scd6-MS2-F on the size distribution of GFP mRNA in polysomes, 80S monosomes, ribosomal subunits, and free mRNPs. Using sedimentation through sucrose density gradients and qRT-PCR analysis of GFP and actin (ACT1) mRNA in the gradient fractions, we reproducibly observed a shift in the fractions with peak abundance of GFP mRNA from polysomes containing 3 to 6 ribosomes (fractions 6–8, 3- to 6-mers) in cells expressing MS2-F alone to those containing 80S monosomes and 2- to 3-mers (fractions 3–5) in cells expressing Scd6-MS2–F (Fig 3A and 3B (P-values for fractions 7 and 4 of 0.031 and 0.0005); S4A and S4B Fig). By contrast, the fractions containing the peak abundance of actin mRNA (fractions 8–10, 5- to 8-mers) did not differ reproducibly between cells expressing Scd6-MS2-F versus MS2–F (Fig 3A & 3C). Despite the shift in peak GFP mRNA abundance from 3- to 5-mers to 2-mers and monosomes, there was no reduction in the proportion of GFP mRNA found in the largest polysomes near the bottom of the gradient on tethering Scd6-MS2-F versus MS–F (Fig 3A and 3B, fractions 10–15). One way to explain these findings is to propose that tethering Scd6-MS2-F inhibits translation to a greater extent at the initiation versus elongation stage of protein synthesis for the fraction of GFP mRNA that shifts towards monosomes and free mRNP, while inhibiting elongation more than initiation on a minority fraction that is retained in heavier polysomes.
(A-C) dcp2Δ transformants from Fig 2A harboring the GFP reporter and expressing Scd6-MS2-F or MS2-F were cultured as in Fig 1B and WCEs were separated by velocity sedimentation on sucrose density gradients and fractionated with continuous monitoring at A254. A representative profile is shown in (A). The abundance of GFP mRNA (B) or ACT1 mRNA (C) was quantitated by RT-qPCR in total RNA extracted from the gradient fractions and plotted as the percentage of total RNA signal in the gradient summed across all fractions. Average results (and ±S.E.M.s) from three biological replicates are shown, which are presented in S4A and S4B Fig. An unpaired Student’s t-test showed that the proportions of mRNA in fractions 7 and 4, representing 4-mers and 1- to 2-mer polysomes, respectively, differed significantly between the Scd6-MS2-F vs. MS2-F transformants, with P-values summarized as: **, P <0.01; *, P <0.05.
Elimination of Ccr4 enhances repression of both GFP mRNA abundance and translational efficiency by tethered Scd6-MS2-F
Having implicated Dcp2 in the reduction of reporter mRNA abundance by tethered Scd6-MS2-F, and noting that mRNA degradation in yeast frequently proceeds via removal of the poly(A) tail followed by decapping , we asked next whether repression of GFP mRNA expression by Scd6-MS2-F requires Ccr4, the major cytoplasmic deadenylase in yeast . Unexpectedly, the reduction in reporter mRNA abundance by Scd6-MS2-F was enhanced rather than diminished in cells lacking Ccr4. We observed a modest (~30%) reduction in GFP mRNA levels in the ccr4Δ mutant containing empty vector or MS2–F (Fig 4B, vector & MS2-F, white vs. grey bars), but tethering Scd6-MS2-F conferred ~3-fold lower GFP mRNA abundance in ccr4Δ vs. WT cells (Fig 4B, Scd6-MS2-F, white vs. grey bars). The resulting >5-fold reduction in mRNA expression by Scd6-MS2-F versus MS2-F seen in the ccr4Δ mutant (Fig 4B, Scd6-MS2-F vs. MS2-F, white bars) is >2-fold larger than that observed in WT cells (Fig 4B, Scd6-MS2-F vs. MS2-F, grey bars; Fig 4C, ΔGFP mRNA, ccr4Δ vs. WT; P = 0.004). These results suggest that Ccr4 is not only dispensable for the reduced abundance of GFP mRNA conferred by tethered Scd6-MS2-F, but actually appears to impede a more efficient repression pathway that can operate in its absence.
(A-D) Transformants of isogenic WT (BY4741) and ccr4Δ (387) strains (A-C), or WT (BY4741) and caf1Δ (7123) strains (D), harboring the GFP reporter and MS2 fusion expression plasmids described in Fig 1 were analyzed for reporter protein (A) and mRNA (B,D) expression as in Fig 1B–1D. Mean values (± S.E.M.s) were determined from at least three biological replicates. Calculations of S.E.M.s for changes in mean ratios of GFP protein and mRNA expression shown in (C & D), and determination of P-values from significance testing of differences in mean values in (A-D) using an unpaired Student’s t-test, were conducted as described in the supporting file S1 Text. P-values are summarized as: **, P <0.01; *, P <0.05. P-values for panel A are <0.0001 (WT) and 0.0012 (ccr4Δ); P-values for panel B are <0.0001 for both WT and ccr4Δ cells.
Interestingly, the absence of Ccr4 also dramatically increased the repression of GFP protein expression by Scd6-MS2–F (Fig 4A, Scd6-MS2-F vs. MS2-F, white vs. grey bars), reducing the Scd6-MS2-F:MS2-F repression ratio for GFP protein from 0.42 to ~0.07 (Fig 4C, ΔGFP protein, ccr4Δ vs. WT; P = 0.0001). Owing to greater repression of GFP protein versus GFP mRNA in ccr4Δ cells, tethering Scd6-MS2-F decreased the TE of GFP mRNA to ~40% of the corresponding TE of ~1.1 observed in WT cells (Fig 4C, ΔTEGFP, ccr4Δ vs. WT; P<0.0001). These findings imply that the presence of Ccr4 also interferes with a more efficient mechanism for translational repression by tethered Scd6-MS2-F that can proceed in ccr4Δ cells. Similar findings were observed on tethering Dhh1-MS2 versus MS2-F to GFP mRNA, as repression of protein expression was greatly enhanced in ccr4Δ versus WT cells (Fig 4A and 4B, Dhh1-MS2-F vs. Scd6-MS2-F, grey vs. white bars), as previously observed with this same tethering system .
Recent findings suggest that the Caf1 subunit of the Ccr4-Not complex cooperates with Ccr4 in the deadenylation and degradation of a subset of yeast mRNAs with low codon optimality, functioning upstream of Dhh1-mediated decapping of such mRNAs . Accordingly, we asked whether eliminating Caf1 from cells would diminish the repression of GFP mRNA abundance conferred by tethered Scd6-MS-F. At odds with this possibility, we observed a greater reduction in GFP mRNA abundance on tethering Scd6-MS2-F in caf1Δ compared to WT cells (Fig 4D; P = 0.01), similar to our findings with the ccr4Δ mutant (Fig 4C). Thus, the Ccr4-Not complex is dispensable for, and even seems to impede, the degradation of mRNAs promoted by tethered Scd6-MS2-F, as observed previously for tethered Dhh1 [6, 34].
Dhh1-dependent translational repression of lacZ reporter mRNA by tethered Scd6-MS2-F is enhanced in the absence of Ccr4
To examine further whether tethered Scd6-MS2-F can repress translation of reporter mRNA independently of Ccr4, we utilized an alternative reporter mRNA, in which the bacterial lacZ gene, encoding β-galactosidase, replaced the GFP coding sequences (Fig 5A), and assays of β-galactosidase activity in cell extracts replaced Western analysis for quantifying reporter protein expression. As observed with the GFP reporter, tethering Scd6-MS2-F conferred an ~2- to 2.5-fold repression of β-galactosidase activity compared to that measured with MS2-F alone in the two different WT strains described above (Fig 5B, Scd6-MS-F vs. MS2-F in WT(BY4741) (P<0.0001) and WT(W303) (P = 0.0005); Fig 5D, grey bars, WT strains). By contrast, β-galactosidase expression on tethering Npl3-MS2-F or Sbp1-MS2-F was indistinguishable from that observed for the corresponding MS2-only controls, or with empty vector in WT cells (S5C Fig). Thus, tethering Scd6-MS2-F, but not the corresponding Npl3 or Sbp1 fusions, confers similar repression of protein expressed from GFP and lacZ reporters. Repression of the lacZ reporter by Scd6-MS2-F was intact in an scd6Δ strain (S5A Fig; P<0.0001), ruling out a contribution of native Scd6 to the function of tethered Scd6-MS2-F. Expression of β-galactosidase activity from heterologous GCN4-lacZ or GAL1-lacZ reporters lacking MS2 binding sites was indistinguishable in cells expressing Scd6-MS-F or MS2–F (S5B Fig), indicating that repression by Scd6-MS-F requires its tethering to lacZ mRNA.
(A) Schema of the MS2CP tethering system, as in Fig 1A, for a lacZ versus GFP reporter mRNA. (B-C) Transformants of WT strains BY4741 or HFY114 (W303 background), as indicated, and ccr4Δ (387), dhh1Δ (3858), or dcp2Δ (CFY1016) strains, containing the MS2-F or Scd6-MS2-F expression plasmids from Fig 1 and lacZ reporter plasmid (pQZ131), were cultured as in Fig 1B. β-galactosidase activities (in units of nanomoles of o-nitrophenyl-b-D-galactopyranoside cleaved per min per mg) were measured in WCEs (B) and lacZ mRNA was quantified by RT-qPCR (C). The ccr4Δ and dhh1Δ strains are isogenic to BY4741; the dcp2Δ mutant is isogenic to HFY114. Mean values (± S.E.M.s) were determined from at least three biological replicates. Calculations of S.E.M.s for changes in mean ratios of β-galactosidase activity and lacZ mRNA expression shown in (D), and determination of P-values from significance testing of differences in mean values in (B-E) using an unpaired Student’s t-test, were conducted as described in the supporting file S1 Text. P-values are summarized as: **, P <0.01; *, P <0.05; n.s., not significant.
Interestingly, lacZ reporter mRNA abundance was not significantly altered, or reduced by only ~20%, by tethered Scd6-MS2-F in the two different WT strains (Fig 5C, Scd6-MS-F vs. MS2-F in WT(BY4741) and WT(W303) (P = 0.02); Fig 5D, ΔlacZ mRNA, WT strains). As a consequence of greater repression of protein versus mRNA expression in WT cells (Fig 5D, WT strains, grey vs. white bars), tethering Scd6-MS2-F reduced the TE of lacZ reporter mRNA by ~30–50% compared to MS2-F alone in the WT strains (Fig 5E, WT strains). Thus, unlike our findings for the GFP reporter (Fig 1F, ΔTEGFP), the ability of tethered Scd6-MS2-F to repress translation of the lacZ reporter was observable in WT cells containing Dcp2.
Tethering Scd6-MS2-F in ccr4Δ cells conferred a dramatic ~9-fold reduction in β-galactosidase expression, and also a ~2.7-fold decrease in lacZ mRNA expression, in comparison to MS2-F alone (Fig 5B and 5C, Scd6-MS-F vs. MS2-F, ccr4Δ; P = 0.001 (panel B), P<0.0001 (panel C). The reductions in β-galactosidase expression and lacZ mRNA on tethering Scd6-MS-F were both greater in ccr4Δ versus WT(BY4741) cells (Fig 5D, grey and white bars, ccr4Δ vs. WT(BY4741); P = <0.0001 (grey), P = 0.0005 (white). However, the reduction in β-galactosidase was relatively larger, yielding an ~3-fold reduction in TE attributable to tethered Scd6-MS2-F, which exceeds the ~2-fold reduction in TE found in WT cells (Fig 5E, ccr4Δ; P = 0.002). Thus, eliminating Ccr4 enhances the repression of both mRNA abundance and translational efficiency of the lacZ reporter by tethered Scd6-MS2-F, as observed above for the GFP reporter (Fig 4C). The finding that tethered Scd6-MS2-F yields a larger reduction in GFP versus lacZ reporter mRNA abundance in WT cells might be explained by proposing that another step in mRNA turnover that is not accelerated by tethered Scd6-MS-F (eg. exonucleolytic degradation by Xrn1 or deadenylation) is more rate-limiting than decapping for the lacZ reporter.
Compared to the levels observed in the isogenic WT strain, the levels of the lacZ reporter mRNA and expression of β-galactosidase were substantially reduced in dhh1Δ cells, even in the presence of empty vector or MS2–F (S5E and S5F Fig, vector and MS2-F, grey vs. white bars). Expression of an unrelated GAL1-lacZ fusion was also diminished by dhh1Δ in cells lacking any MS2 proteins, albeit to a smaller degree (S5D Fig). While these effects of dhh1Δ complicated our analysis, we nevertheless obtained results consistent with the earlier conclusion that Dhh1 is required for translational repression by tethered Scd6-MS2-F. Despite the general reductions in lacZ mRNA levels in dhh1Δ cells, Scd6-MS2-F conferred ~2.5-fold lower mRNA levels versus MS2-F alone in this mutant (Fig 5C, Scd6-MS2-F vs. MS2-F, dhh1Δ; P = 0.0001). Thus, eliminating DHH1 uncovers a reduction in lacZ mRNA abundance on tethering Scd6-MS2-F not observed in the isogenic WT strain (Fig 5D, white bars, dhh1Δ vs. WT; P = 0.0002). Expression of β-galactosidase in dhh1Δ cells also showed an ~2-fold reduction on tethering Scd6-MS2-F versus MS2–F (Fig 5B, Scd6-MS2-F vs. MS2-F, dhh1Δ; P<0.0001), as in the WT strain (Fig 5D, white grey bars, dhh1Δ vs WT). Because tethered Scd6-MS2-F reduced both reporter mRNA and reporter protein by ~60% compared to MS2-F alone (Fig 5D, dhh1Δ), it did not confer any reduction in TE of lacZ mRNA, in contrast to the ~50% reduction in TE observed in WT cells (Fig 5E, dhh1Δ vs. WT; P = 0.001), which is consistent with Dhh1 being required for translational repression of the lacZ reporter.
Deletion of DCP2 led to an ~2.5-fold increase in lacZ mRNA levels in the presence of MS2-F alone (Fig 5C, MS2-F, dcp2Δ vs. WT(W303)), which also occurred in the presence of vector alone and, hence, is not a consequence of tethering (S5G Fig). Tethering Scd6-MS2-F evoked an ~27% additional increase in lacZ reporter mRNA versus tethering MS2-F in dcp2Δ cells (Fig 5C, Scd6-MS2-F vs. MS2-F, dcp2Δ; P = 0.03), eliminating the small reduction in lacZ mRNA abundance on tethering Scd6-MS2-F in the isogenic WT strain noted above (Fig 5C, Scd6-MS2-F vs. MS2-F, WT; Fig 5D, white bars, dcp2Δ vs. WT; P = 0.001). The fact that the ~1.3-fold increase in lacZ mRNA conferred by tethered Scd6-MS2-F versus MS2-F in dcp2Δ cells is smaller than the corresponding ~2-fold increase in GFP mRNA abundance conferred by tethered Scd6-MS2-F in dcp2Δ versus WT cells shown above (Fig 2C, black vs. dark grey bars) might reflect that tethering Scd6-MS2-F confers a much smaller reduction in lacZ mRNA (~1.1-fold) versus GFP mRNA abundance (~2.6-fold) in WT cells (Figs 5D vs. 2D, WT cells, white bars). Coupling the small increase in lacZ mRNA abundance with a slight reduction in β-galactosidase expressed on tethering Scd6-MS2-F versus MS2-F in dcp2Δ cells (Fig 5B and 5C, Scd6-MS2-F vs. MS2-F, dcp2Δ), results in an ~30% reduction in TE compared to tethering MS2-F alone, which is indistinguishable from that observed in the isogenic WT strain (Fig 5E, dcp2Δ vs. WT(W303)). These findings are consistent with our conclusion above that Dcp2 is dispensable for translational repression by tethered Scd6-MS2-F.
In summary, tethering Scd6-MS2-F to the lacZ reporter confers a decrease in translational efficiency that appears to be dependent on Dhh1, independent of Dcp2, and dampened by Ccr4; and Ccr4 also diminishes the repression of lacZ mRNA abundance by tethered Scd6-MS2-F. All of these observations are in agreement with our findings for the GFP reporter. Unlike our results for the GFP reporter, where deleting DCP2 reduced the TE on tethering Scd6-MS2–F (Fig 2E, cols. 1–2), dcp2Δ had little effect on TE of the lacZ reporter because it did not substantially reduce the apparent degradation of lacZ mRNA by tethered Scd6-MS2-F, conferring only a small increase in lacZ mRNA abundance (Fig 5D, WT vs. dcp2Δ, white bars.)
The LSm but not the RGG domain of Scd6 is required for repression of reporter expression by tethered Scd6-MS2-F
Scd6 interacts with eIF4G via the C-terminal region of Scd6 containing the RGG domain . Interaction partners of the N-terminal LSm domain of Scd6 are unknown; however, the LSm domains in Scd6 homologs from different species mediate binding to Dcp2 in S. pombe , both Dcp1 and the translational repressor CUP in D. melanogaster , and decapping activators and translational repressors 4E-T and EDC4 in humans . We examined the importance of the LSm and RGG domains of Scd6 for reporter mRNA repression by truncating the Scd6-MS2-F fusion at the N- or C-terminal ends to remove these domains individually (Fig 6A). Eliminating the LSm domain completely abrogated repression of both protein and mRNA expressed from the GFP reporter by tethered Scd6-MS2–F (Fig 6D and 6E, white vs. grey bars; P = 0.0001 (D), P<0.0001 (E) for ΔLSm-Scd6-MS2-F vs. Scd6-MS2-F), without detectably altering expression of the Scd6-MS2-F fusion protein (Fig 6B, upper blot, lanes 5–12). The fact that no reduction in GFP mRNA occurs on tethering the ΔLSm-Scd6-MS2-F variant implies that the LSm domain is required for accelerated mRNA turnover conferred by WT tethered Scd6-MS2-F. Because there is no reduction in GFP protein expression on tethering the ΔLSm-Scd6-MS2-F, despite high levels of the GFP reporter mRNA, we can also infer that translational repression of GFP mRNA is eliminated by removing the LSm domain. Repression of β-galactosidase expression from the lacZ reporter was also abolished by removing the LSm domain from Scd6-MS2–F (Fig 6G, white vs. grey bars; P<0.0001 for ΔLSm-Scd6-MS2-F vs. Scd6-MS2-F). Because there is little or no reduction in lacZ mRNA abundance on tethering WT Scd6-MS2-F, it seems likely that translational repression is abrogated by removing the LSm domain for this reporter mRNA as well. (However, we cannot discard the unlikely possibility that tethering ΔLSm-Scd6-MS2-F would substantially increase the abundance of lacZ mRNA and thereby mask efficient translational repression by the ΔLSm variant.)
(A) Diagrams representing the domain organization of full-length Scd6 (Scd6-MS2-F) or variants lacking amino acids 1–83 at its N-terminus (ΔLSm-Scd6-MS2-F) or amino acids 286–312 at its C-terminus (ΔRGG-Scd6-MS2-F), present in the corresponding fusions to MS2-F. MS2 and FLAG tags are not depicted. (B-F) Transformants of WT strain BY4741 containing the GFP reporter plasmid pJC429 and expression plasmids for MS2-F (pQZ130) and Scd6-MS2-F (pQZ127), and either ΔLSm-Scd6-MS2-F (pQZ139) (B, D-E) or ΔRGG-Scd6-MS2-F (pQZ142) (C & F) were analyzed for GFP protein (B-D & F) and mRNA (E) expression as in Fig 1B–1D. (G) Transformants of WT strain BY4741 harboring the lacZ reporter plasmid pQZ131 and the expression plasmids for MS2-F, Scd6-MS2-F, or ΔLSm-Scd6-MS2-F used in (B) were analyzed for β-galactosidase expression as in Fig 5B. Mean values (± S.E.M.s) were determined from at least four biological replicates. Determination of P-values from significance testing of differences in mean values in (D-G) using an unpaired Student’s t-test, were conducted as described in the supporting file S1 Text. P-values are summarized as: **, P <0.01; *, P <0.05.
In contrast to our findings on removing the LSm domain, eliminating the RGG domain from Scd6-MS2-F had no apparent effect on repression of the GFP reporter by Scd6-MS2–F (Fig 6C, GFP blot; Fig 6F, black vs. grey; P = 0.002), although an apparent increase in expression of the ΔRGG-Scd6-MS2-F versus WT Scd6-MS2-F fusion (Fig 6C, upper blot, lanes 5–12) might have obscured a reduced efficiency of reporter repression for the ΔRGG variant. These findings indicate that the LSm domain, and most likely interactions it mediates with effector proteins, is required for both enhanced degradation of GFP reporter mRNA and translational repression of both reporters by tethered Scd6-MS2-F. By contrast, interaction of Scd6-MS2-F with eIF4G via the Scd6 RGG domain might be dispensable for translational repression when Scd6 is tethered tightly to the mRNA; although we cannot eliminate the possibility that translational repression is impaired by the ΔRGG truncation and that the efficient repression of GFP reporter protein expression conferred by ΔRGG-Scd6-MS2-F occurs exclusively from accelerated mRNA turnover.
Scd6 and Dhh1 cooperate in regulating mRNA abundance and translational efficiencies of particular native mRNAs in vivo
To determine whether Scd6 and Dhh1 participate in regulating the abundance or translation of native yeast mRNAs, we conducted ribosome footprint profiling and RNA-Seq analyses on the WT, dcp2Δ, scd6Δ and dhh1Δ strains in the W303 genetic background, cultured in rich (YPD) medium. We also analyzed isogenic dcp2Δscd6Δ and dcp2Δdhh1Δ double mutants, anticipating that changes in translational efficiency might be more evident in the absence of mRNA decapping by Dcp2. Independent RNA-Seq analysis was also conducted in parallel on two isogenic scd6Δ strains, an additional isogenic dhh1Δ strain and isogenic mutants lacking the Dcp2-decapping activators Pat1 and Lsm1  to determine whether Pat1 and Lsm1 contribute to Scd6-mediated repression of mRNA abundance. The results of biological replicates were highly correlated for both ribosome-protected fragments (RPFs) and mRNA sequences for all strains analyzed (S6A–S6L Fig).
RNA-Seq analysis of WT and scd6Δ strains identified 83 mRNAs whose abundance was significantly up-regulated in the mutant by ≥1.4-fold at an FDR of <0.01, with a median fold-change (FC) compared to WT of ~1.8, which is significantly higher than the median FC for all mRNAs (which is 1.0 (log2(ΔmRNA) = 0)) owing to normalization for equal RNA read numbers for all genes in each strain) (Fig 7A, scd6Δ). Interestingly, this group of mRNAs also displayed significantly increased abundance in the isogenic dhh1Δ, pat1Δ, and lsm1Δ strains, with median FCs of ~3.3, ~1.9, and ~1.8, respectively (Fig 7A, cols. 2–4). Consistent with the latter, most of the 83 mRNAs up-regulated in scd6Δ cells are a subset of the larger group of 733 mRNAs elevated to the same degree in dhh1Δ cells (Fig 7B). Importantly however, derepression of these 83 mRNAs was not observed on deleting SCD6 or DHH1 in the strain lacking DCP2, ie. when comparing the dcp2Δscd6Δ and dcp2Δdhh1Δ double mutants to the dcp2Δ single mutant (Fig 7A, rows 5–6). These findings are consistent with the notions that: (i) Scd6 accelerates degradation of a subset of native mRNAs, (ii) that Dhh1, Pat1, and Lsm1 all participate in this down-regulation of mRNA abundance, and (iii) that the decapping enzyme subunit Dcp2 is required for both Scd6- and Dhh1-enhanced degradation of the set of Scd6-targeted mRNAs.
(A) Notched box-plots of log2 mRNA changes (ΔmRNA) in the indicated mutants for 83 mRNAs exhibiting ≥1.4-fold increased mRNA abundance in scd6Δ versus WT cells (at FDR<0.01). The 83 mRNAs were identified by RNA-Seq analysis of scd6Δ strains (SYY2352 and SYY2353) and isogenic WT strain (HFY114); and the RNA changes shown for the mutants were determined by RNA-Seq analysis of isogenic strains of the indicated genotypes: dhh1Δ (SYY2686), pat1Δ (SYY2674), lsm1Δ (SYY2680), all compared to WT strain (HFY114); and dcp2Δscd6Δ (FZY843) and dcp2Δdhh1Δ (QZY128) compared to the dcp2Δ strain (CFY1016). (In this and all subsequent plots, the double mutants carry an (*) to indicate that their values have been compared to the dcp2Δ single mutant rather than to WT.) Non-overlapping notches indicate that the true medians of the two groups are different with a 95% confidence level. (B) Overlap between 83 mRNAs exhibiting ≥1.4-fold increased mRNA abundance in scd6Δ (SYY2352 and SYY2353) vs. WT (HFY114) cells and 733 mRNAs similarly up-regulated in dhh1Δ cells (QZY126), both at FDR<0.01. (C) Notched box-plots of log2 TE changes (ΔTE) in the indicated mutants for 78 mRNAs exhibiting ≥1.33-fold increased ribosome abundance across the CDS in scd6Δ versus WT cells (at P≤0.01). The 78 mRNAs were identified by ribosome profiling of scd6Δ strain (SYY2353); and TE changes were calculated from ribosome profiling/RNA-seq data conducted on this scd6Δ strain, dhh1Δ strain (QZY126, dhh1Δ(z)), and WT strain (HFY114); and on the dcp2Δscd6Δ, dcp2Δdhh1Δ and dcp2Δ strains mentioned in (A). TE changes for the dhh1Δ(r) and dhh1Δ(j) strains in the BY4743 background were calculated from published ribosome profiling/RNA-Seq data from Radhakrishnan et al.  and Jungfleisch et al. , respectively. (D) Overlap between 78 mRNAs exhibiting ≥1.33-fold increased ribosome occupancy (p<0.01) in scd6Δ (SYY2353) vs. WT (HFY114) cells and 968 mRNAs similarly up-regulated by ≥1.4-fold (FDR<0.01) in dhh1Δ cells (QZY126). (E) Notched box-plots of log2(ΔTE) values in the indicated mutants for 53 mRNAs exhibiting ≥1.33-fold increased TE in scd6Δ versus WT cells (at P≤0.1), identified by ribosome profiling/RNA-Seq analysis of scd6Δ (SYY2353) and WT (HFY114) strains. The TE changes were calculated from the data sets described in (B). (F) Overlap between 53 mRNAs exhibiting ≥1.33-fold increased TE (p<0.10) in scd6Δ (SYY2353) vs. WT (HFY114) cells and 283 mRNAs similarly up-regulated by ≥1.33-fold (FDR<0.10) in dhh1Δ cells (QZY126). P-values in (B-F) were assigned based on the hypergeometric distribution. For the boxplots in panels A, C, & E, the changes in mRNA abundance or TE for the relevant group of mRNAs found in each of the indicated mutants were plotted irrespective of whether the changes exhibit statistical significance in that mutant, to allow a coherent comparison of the behavior of the complete cohort of mRNAs across the entire panel of mutants. Statistical significance is evaluated for differences in the median changes found in the different mutants, with non-overlapping notches indicating with 95% confidence that the median changes found for two mutants differ from one another.
We obtained complementary results for a group of 346 mRNAs whose abundance was significantly increased (at FDR<0.01) in an isogenic dhh1Δ mutant, exhibiting an ~3-fold increase in median mRNA abundance in dhh1Δ vs. WT cells (S7A Fig, dhh1Δ(z)), and also showing ~1.9-fold and ~1.8-fold increases in two published RNA-seq datasets for a dhh1Δ mutant in the BY4741 background [10, 37] (S7A Fig, dhh1Δ(r) and dhh1Δ(j)). This group of Dhh1 down-regulated mRNAs also displays a slight, but statistically significant, ~15% derepression in the scd6Δ mutant (S7A Fig, scd6Δ). Supporting these findings, hierarchical clustering analysis of expression changes for all mRNAs revealed that a large proportion of genes exhibit altered mRNA levels in the same direction in response to scd6Δ or dhh1Δ (S7B Fig), which is particularly evident for the mRNAs showing the largest fold-changes in dhh1Δ vs. WT cells (S7C Fig), with the magnitudes of these changes being generally greater in dhh1Δ vs. scd6Δ cells (S7B and S7C Fig). These results suggest that Scd6 contributes appreciably to Dhh1-enhanced degradation of a large fraction of the mRNAs whose abundance is repressed by Dhh1. As observed for the Scd6 down-regulated mRNAs, the derepression of mRNA levels for the group of 346 Dhh1-repressed mRNAs conferred by either scd6Δ or dhh1Δ was eliminated when these mutations were made in cells lacking DCP2 (S7A Fig, cf. cols. 1–2 and 5–6), indicating that Dcp2 is required for robust Dhh1-mediated mRNA turnover.
The two groups of 83 and 346 mRNAs whose abundance is derepressed in scd6Δ (Fig 7A) or dhh1Δ cells (S7A Fig), respectively, were interrogated next for changes in translation efficiency (TE) in different mutants by combining the results of ribosome profiling and RNA-Seq experiments conducted on the same cell cultures. The TE of each mRNA was calculated as the sum of RPFs divided by the sum of RNA reads across the CDS, and the change in TE (ΔTE) was calculated as the ratio of TE in mutant versus WT cells. (Because RPFs and mRNA reads for each gene are normalized to RPF or mRNA reads for all genes in each strain, the ΔTE for each gene is determined relative to the median ΔTE for all genes, which is ~1.0). Both groups of mRNAs showing derepression of mRNA abundance in scd6Δ or dhh1Δ cells also exhibited modest increases in median TE in our dhh1Δ mutant of ~12–15% (S7D and S7E Fig, col. 5 in each panel). Comparable or somewhat greater increases in TE were identified in the two published ribosome profiling/RNA-seq datasets for a dhh1Δ mutant in the BY4741 background [10, 37] (S7D and S7E Fig, cols. 3–4 in each panel); whereas the scd6Δ mutation increased the median TE for these groups of mRNAs by only ~5% (S7D and S7E Fig, col. 1). Despite the modest TE increases for these groups of mRNAs conferred by dhh1Δ, it is noteworthy that these changes were not observed on comparing the dcp2Δdhh1Δ double mutant to the dcp2Δ single mutant (S7D and S7E Fig, cols. 5–6 in each panel), providing genetic evidence that the TE changes are genuine, and indicating that translational repression by Dhh1 is dependent on Dcp2.
Evidence for Dcp2-dependent translational repression of native mRNAs by Scd6 was provided by examining a group of 78 mRNAs exhibiting significantly increased ribosome occupancies in the scd6Δ mutant, and for a second group of 53 mRNAs showing the largest TE increases conferred by scd6Δ. In response to scd6Δ, both groups of mRNAs exhibit increased median TEs of ~1.2- and ~1.5-fold, respectively, in otherwise WT cells, but not in the presence of dcp2Δ (Fig 7C & 7E, cf. cols. 1–2 in each panel). Comparable, or somewhat greater, increases in median TE were observed for both groups of mRNAs in all three dhh1Δ datasets (Fig 7C & 7E, cols. 3–5), indicating that Dhh1 contributes to translational repression of a substantial proportion of the mRNAs thus repressed by Scd6. This last inference is further supported by the significant overlaps between mRNAs exhibiting increased ribosome occupancies or TEs in response to scd6Δ and the larger groups of mRNAs showing comparable increases in ribosome occupancy or TE in response to dhh1Δ (Fig 7D & 7F). Moreover, clustering analysis for all mRNAs showed that the majority of mRNAs displaying increased TEs in response to scd6Δ also exhibit TE increases of generally greater degree in response to dhh1Δ; although it is also evident that many mRNAs translationally repressed by Dhh1 are not repressed by Scd6 (Fig 8A and 8B). Once again, comparing the dcp2Δdhh1Δ double mutant to the dcp2Δ single mutant revealed that dcp2Δ largely suppresses the TE increases conferred by dhh1Δ in DCP2 cells (Fig 7C & 7E, cf. cols. 5–6 in each panel), supporting a widespread requirement for Dcp2 in translational repression by Dhh1.
(A-B) Hierachical clustering analysis conducted with the R heatmap.2 function from the R gplots library, using the default “hclust” hierarchical clustering algorithm, using ribosome profiling data from scd6Δ (SYY2353), dhh1Δ (QZY126, dhh1Δ(z)), and WT (HFY114) strains. Approximately 50 genes were removed for which no data were available in one of the strains, or where the log2(ΔTE) value was >4 or <-4 in one of the mutant vs. WT comparisons, after which separate clustering analysis was performed on two sets of mRNAs in which all log2(ΔTE) values fell between -2 or +2 (panel A, 5276 mRNAs), or in which the log2(ΔTE) value in one of the mutants was < -2 or > +2 (panel B, 45 mRNAs). The color key for log2(ΔTE) values is indicated above each analysis. (C) Exemplar gene exhibiting increased TE in both scd6Δ and dhh1Δ versus WT cells. Integrated Genomics Viewer (Broad Institute) display of ribosome-protected fragments (RPFs) and mRNA reads across the YOR173W gene from two biological replicates each for WT, scd6Δ and dhh1Δ strains, shown in units of rpkm (reads per 1000 million mapped reads). Position of the CDS (magenta) is at the bottom with the scale in bp; scales of rpkm for each track are on the left, and calculated ΔRPF, ΔmRNA and ΔTE values between each mutant and WT are on the right. (D-E) Boxplot analysis of changes in ribosome occupancy or TE versus Dhh1 occupancy. Dhh1 RIP-seq enrichment values from Miller et al (2018) were equally divided into five pentiles of 739 genes from lowest to highest values and plotted against the log2(Δribo) values (D) or log2(ΔTE) values (E) determined by ribosome profiling analysis of scd6Δ strain SYY2353 and WT strain HFY114. The Pearson correlation coefficients for the relationship between log2(Δribo) values (panel D) or log2(ΔTE) values (panel E) and Dhh1 enrichment for all mRNAs are 0.2 (P = 2 X 10−34) and 0.16 (P = 2 X 10−22), respectively.
The mRNAs encoded by YOR173W, YHR033W, YGR088W, YFR017C, and YNR034W-A exhibit TE increases conferred by scd6Δ, and they respond similarly to dhh1Δ, while exhibiting larger increases in mRNA and TE levels in dhh1Δ versus scd6Δ cells (Fig 8C and S8A–S8D Fig). Gene-ontology (GO) analysis revealed that mRNAs whose transcript abundance was derepressed in dhh1Δ or scd6Δ cells are enriched in related functional categories of genes involved in metabolism of energy reserves and other aspects of carbohydrate metabolism; and the Dhh1-repressed mRNAs are also enriched in stress-response genes (S9A and S9B Fig). These results suggest that Scd6 and Dhh1 cooperate in repressing both mRNA abundance and translational efficiency for a discrete subset of native mRNAs in nutrient-replete medium.
To provide evidence supporting a direct role for Dhh1 in translational repression of native mRNAs by Scd6, we interrogated published results from deep-sequencing of mRNAs specifically immunoprecipitated with epitope-tagged Dhh1 (RIP-Seq) from WT cells grown on rich medium . The 3686 mRNAs for which both RIP-Seq and ribosome profiling data exist were sorted into 5 equal percentiles based on Dhh1 enrichment values in RIP-seq and compared for translation changes in response to dhh1Δ or scd6Δ. This analysis revealed a direct correlation between Dhh1 enrichment values and changes in both ribosome occupancies and TEs in response to dhh1Δ (S10A and S10B Fig), and a similar correlation exists between Dhh1 enrichment and changes in mRNA levels in dhh1Δ vs. WT cells (S10C Fig), as noted previously . These correlations support previous conclusions that Dhh1 binding to mRNAs is associated with accelerated mRNA decay and translational repression. Interestingly, Dhh1 enrichment values are likewise correlated with changes in ribosome occupancies and TEs in response to scd6Δ (Fig 8D and 8E), supporting the notion that Scd6 translational repression of many native mRNAs involves recruitment of Dhh1. Finally, we considered the possible contribution of codon optimality in dictating susceptibility of mRNAs to Scd6. Previously, low codon optimality was associated with Dhh1-mediated mRNA decay in part by demonstrating an inverse correlation between the sTAI value, a measure of overall codon optimality of the mRNA , and the change in mRNA abundance in dhh1Δ versus WT cells . This correlation is also evident in our dhh1Δ dataset and that of Jungfleisch et al.  (S11B and S11C Fig), although less pronounced than observed in the data from Radhakrishnan et al.  (S11A Fig). A similar, modest trend was also evident for mRNA changes observed here in scd6Δ cells (S12A Fig), suggesting that mRNAs exhibiting Scd6-dependent mRNA degradation have a tendency to exhibit poor codon optimality. Interestingly, however, the group of 82 mRNAs whose abundance is most strongly derepressed in scd6Δ cells (characterized in Fig 7A) exhibit sTAI values that are somewhat higher, not lower, than the genome average value (S12B Fig), indicating that poor codon optimality is not the key determinant of Scd6-dependent mRNA turnover for the transcripts that it most strongly represses.
In this report we have shown that tethering an Scd6-MS2-F fusion protein to two different reporter mRNAs harboring MS2 binding sites represses reporter protein expression and, in the case of the GFP reporter, also reduces reporter mRNA abundance. Together with Npl3 and Sbp1, Scd6 is one of three yeast proteins containing RGG domains capable of binding to the C-terminal domain of eIF4G and repressing translation initiation in cell extracts . However, we observed no effects on reporter expression on tethering Npl3 or Sbp1. The fact that tethered Scd6-MS2 reduced β-galactosidase expression without reducing lacZ mRNA abundance implied a reduction in translational efficiency of the lacZ mRNA. While this inference was not possible for the GFP reporter in WT cells, owing to comparable reductions in protein and mRNA expression, it was clearly indicated by the much greater repression of GFP protein versus GFP mRNA conferred by tethered Scd6-MS2 in the ccr4Δ mutant. Moreover, translational repression of the GFP reporter was revealed in the dcp2Δ mutant, as the reduction in GFP mRNA abudance was diminished while repression of GFP protein was maintained. Thus, we propose that tethering Scd6-MS2 evokes translational repression of both GFP and lacZ reporter mRNAs, and also degradation of the GFP reporter mRNA, with the latter dependent on decapping by Dcp2. Additional evidence for translational repression was provided by our finding that tethering Scd6-MS2 in dcp2Δ cells shifted a proportion of the GFP mRNA from large to smaller polysomes and monosomes, suggesting a reduced rate of translation initiation.
We implicated Dhh1 in translational repression of both GFP and lacZ mRNAs, but found it to be dispensable for the degradation of GFP mRNA, evoked by tethered Scd6-MS2-F. Thus, the TE values for both GFP and lacZ mRNAs were higher in dhh1Δ versus WT cells (Figs 2E & 5E); and also were higher in the dhh1Δ dcp2Δ double mutant compared to the dcp2Δ single mutant for the GFP reporter (Fig 2E). Importantly, repression of both GFP protein and GFP mRNA abundance by tethered Scd6-MS2 is absent in the dhh1Δ dcp2Δ double mutant, whereas GFP protein repression is intact in dcp2Δ cells, and repression of GFP mRNA abundance occurs in dhh1Δ cells (Fig 2D). These comparisons indicate that Dcp2 is required for efficient mRNA degradation while Dhh1 is required for full translational repression of GFP reporter mRNA. The role of Dhh1 in translational repression is further supported by our finding that tethering Dhh1 as an MS2 fusion represses expression of GFP protein more than GFP mRNA abundance in ccr4Δ cells, as observed previously ), similar to the effects of tethered Scd6-MS2–F (Fig 4A and 4B). These results are consistent with previous demonstrations of direct interactions between Scd6 and Dhh1 [23, 24]. Our finding that repression of GFP mRNA abundance by Scd6-MS2 remains intact in the dhh1Δ strain implies that Dhh1 is not required for recruitment or activation of Dcp1/Dcp2 on this reporter mRNA, which could involve instead the known direct interaction of Scd6 with Dcp2 [7, 23, 24, 27, 29].
Although we could readily observe that tethered Scd6-MS2-F reduces the TE of the GFP reporter mRNA in ccr4Δ cells, it was not possible to infer translational repression in WT cells because tethered Scd6-MS2-F repressed GFP reporter and protein expression almost equally. However, translational repression by tethered Scd6-MS2-F was uncovered in the dcp2Δ strain in which the accelerated degradation of GFP reporter mRNA was diminished. The ability to observe translational repression after uncoupling it from mRNA turnover in a dcp2Δ mutant has been reported previously for Dhh1 using the same tethering assay and GFP reporter employed here , and also in similar experiments involving the tethering of Dhh1 to reporter mRNA in mutant strains where mRNA turnover was impaired by elimination of Dcp1 or Xrn1 . Together, these findings indicate that both Scd6 and Dhh1 can repress translation independently of their functions in activating mRNA decay. In addition to the genetic uncoupling of mRNA decay from translational repression accomplished in yeast, these processes have been kinetically resolved in miRNA-mediated repression in Drosophila cells  and zebrafish  by showing that translational repression precedes mRNA turnover.
Previous findings showed that tethered Dhh1-MS2 interferes with the elongation stage of translation and is associated with the presence of slowly decoded suboptimal codons in the reporter mRNA . Current evidence suggests that Dhh1 can be recruited to mRNAs by slowly elongating ribosomes and triggers decapping and subsequent mRNA degradation; and that Dhh1 can also impede the progression of 80S ribosomes at suboptimal codons, at least when tethered to mRNA or overexpressed in cells . These findings are ostensibly at odds with our conclusion that Dhh1 participates in translational repression by tethered Scd6-MS2-F and the results of our polysome analysis indicating that tethered Scd6-MS2 does not shift the GFP reporter mRNA into larger polysomes (Fig 3), which would be expected for slower elongation. Rather, tethered Scd6-MS2 appears to shift the mRNA towards smaller polysomes and possibly free mRNP (Fig 3). However, Dhh1 can bind directly to both 40S and 60S subunits [6, 10], and has been implicated in the inhibition of bulk translation initiation during carbon starvation  or when overexpressed in nutrient-replete cells . Moreover, Dhh1 can inhibit 48S PIC assembly when added to cell extracts . It has been suggested that Dhh1 can inhibit either initiation by interacting with the PIC, or elongation by binding to translating 80S ribosomes, and the relative importance of these mechanisms could vary with the mRNA, depending, for example, on the number and position of suboptimal codons [6, 10]. Although tethered Dhh1-MS2 was found to inhibit elongation on the GFP reporter mRNA , for which tethered Scd6-MS2 appears to have a relatively greater effect on initiation, perhaps the amount of Dhh1 that would be recruited to the reporter by Scd6-MS2 is lower than achieved by tethering Dhh1-MS2 itself, and may be sufficient to inhibit initiation but not elongation. As shown for Dhh1, Scd6 can also inhibit 48S PIC formation in cell extracts [7, 22] and tethered Scd6-MS2-F might work in conjunction with Dhh1 to produce a rate-limiting initiation defect on the GFP reporter.
We found that the LSm domain of Scd6 is indispensable for the ability of tethered Scd6-MS-F to repress GFP reporter mRNA and protein expression (Fig 6), implying its requirement for both translational repression by Dhh1 and decapping by Dcp2. Whereas the LSm domains of Scd6 homologs in S. pombe and D. melanogaster have been shown to interact with Dcp1 or Dcp2, the interactions with Dhh1 homologs involve the DFDF and TFG domains in the C-terminal regions of Scd6 homologs in D. melanogaster  and humans . If the LSm domain in S. cerevisiae Scd6 likewise interacts with Dcp1 or Dcp2, this could explain the requirement for this domain in stimulating mRNA degradation by tethered Scd6-MS2-FL; however, the additional requirement of the LSm domain for translational repression presumably does not involve direct recruitment of Dhh1. Considering that the LSm domains in Scd6 homologs also bind the translational repressor proteins CUP in Drosophila  and 4E-T in humans , we suggest that this domain in S.cerevisiae Scd6 likewise recruits an additional repressor protein that, together with Dhh1, mediates translational inhibition by tethered Scd6-MS2-FL.
In contrast to our findings on the LSm domain of Scd6, we found that the RGG domain at the C-terminus of Scd6 was not needed for repression of the GFP reporter by tethered Scd6-MS-F. As interaction of the Scd6 RGG domain with eIF4G was found previously to be required for the inhibition of translation initiation by Scd6 in cell extracts , it is possible that this interaction interferes with the intrinsic function of the eIF4G C-terminal region in 48S PIC assembly. Alternatively, the Scd6-RGG/eIF4G interaction could serve primarily to recruit Scd6 to eIF4F-mRNP complexes for inhibition of 43S PIC recruitment, via Scd6 interactions with other components of the eIF4F-mRNP or 43S PIC, or by recruiting repressor proteins like Dhh1 or Pat1 to do so. Although our findings are more consistent with the latter possibility, an inhibitory interaction of the Scd6 RGG domain with the eIF4G C-terminus might still be crucial for translational repression on native mRNAs to which Scd6 is not tightly tethered.
Our results using the tethering assay demonstrate that Scd6 can repress both mRNA abundance and translational efficiency of specific reporter mRNAs when tethered to these mRNA targets in vivo. Using RNA-Seq and ribosome profiling we went on to provide evidence that Scd6 is involved in repressing the abundance and/or translation of a discrete set of native yeast mRNAs in cells cultured in rich medium. The abundance of a group of 83 mRNAs was significantly up-regulated in scd6Δ cells, and the functions of the encoded proteins are enriched in the processes of metabolism of energy reserves and other aspects of carbon metabolism. Similarly, the abundance of a group of 346 mRNAs was derepressed in dhh1Δ cells, which are enriched for the same functional categories, as well as in stress response functions. These findings are consistent with recent results indicating that Dhh1-occupied mRNAs are enriched for transcripts whose levels are derepressed in cells depleted of glucose or a preferred nitrogen source . Importantly, the group of 83 mRNAs whose levels are elevated in scd6Δ cells also tend to be elevated in mutants lacking Dhh1, Pat1, or Lsm1 (Fig 7A and 7B), suggesting cooperation among these decapping activators in degradation of many native Scd6 target mRNAs. The groups of mRNAs whose abundance is derepressed in scd6Δ or dhh1Δ cells also exhibit a modest up-regulation in median TE values in dhh1Δ cells (S7D and S7E Fig), consistent with concerted mRNA destabilization and translational repression by Dhh1 on a subset of these mRNAs. It is possible that the observable extent of translational repression of these mRNAs is dampened by their accelerated degradation, in the manner we observed for the GFP reporter mRNA on tethering Scd6-MS2-F.
For two additional groups of mRNAs exhibiting the largest increases in ribosome occupancy or TE in scd6Δ cells, we again observed a contribution of Dhh1 to translational repression (Fig 7C–7F), similar or even greater in magnitude to that of Scd6 for these groups of mRNAs (Fig 7C & 7E). Broad cooperation between Scd6 and Dhh1 in translational control was also evident in genome-wide comparisons of TE changes in scd6Δ vs. dhh1Δ cells (Fig 8A and 8B). Moreover, we found that Dhh1 occupancy is correlated with increased translation and increased TE in scd6Δ cells (Fig 8D and 8E) as well as in dhh1Δ cells (Fig 9A and 9B). These findings support our conclusion reached from tethering assays that translational repression by Scd6 involves Dhh1.
(i) Closed-loop mRNP formation by mutual interactions of eIF4G with eIF4E bound to the mRNA cap and PABP bound to the poly(A) tail of mRNA, activating mRNA for translation initiation. (ii) Scd6 is recruited to the mRNA by binding to the C-terminus of eIF4G, but might also interact independently with the 3’UTR. (iii) Scd6 recruits Dhh1 and Dcp1/Dcp2 to form an inactive mRNP incapable of recruiting the 43S PIC complex near the 5’ end of the mRNA. (iv) Dcp1/Dcp2 decaps the mRNA, dissociating eIF4E, and exposing the 5’ end of the mRNA for subsequent exonucleolytic degradation (depicted as dotted line). (v) Recruitment of the Ccr4/Not complex to the mRNA interferes with Scd6-mediated translational repression and mRNA degradation.
Interestingly, our analyses of double mutants lacking Dcp2 in addition to Scd6 or Dhh1 indicated that translational repression, as well as mRNA degradation, mediated by Scd6 or Dhh1 is highly dependent on Dcp2 for most native mRNAs regulated by these proteins. Dcp2-dependence was expected for repression of mRNA levels, as decapping is an established prelude to mRNA degradation by the 5’-3’ exonuclease Xrn1 in yeast . We did not anticipate a requirement for Dcp2 in translational repression, however, as translational repression of the GFP reporter by both tethered Scd6-MS-F (Fig 2) and Dhh1-MS  was uncovered in dcp2Δ cells by the reduced GFP mRNA turnover, rather than being diminished. One way to account for this discrepancy is to propose that, on native mRNAs targeted by Scd6 or Dhh1, Dcp2 is required for stable assembly of a translation repression complex capable of impeding 43S PIC association (Fig 9, (iii)), in addition to decapping mRNA to enhance degradation (Fig 9, (iv)), and this contribution of Dcp2 to translational inhibition is bypassed by artificially increasing the occupancies of Scd6 or Dhh1 on the mRNAs via tethering. It is also possible that a broad effect of dcp2Δ in increasing the abundance of many capped mRNAs, possibly with shortened poly(A) tails, indirectly diminishes translational repression by Dhh1 or Scd6 binding to target mRNAs. Although the underlying mechanism for the role of Dcp2 in translational repression of native mRNAs remains to be determined, the fact that dcp2Δ completely suppressed the increased mRNA levels and TE values conferred by scd6Δ or dhh1Δ (Fig 7A, 7C and 7E) provides genetic evidence that, while modest in magnitude for scd6Δ, these changes are physiologically relevant for the affected mRNAs.
An unexpected finding from the tethering assays is that Scd6-MS2 binding conferred only a small decrease in lacZ reporter mRNA levels, which was limited to one genetic background (Fig 5C), but a marked reduction in abundance of GFP mRNA (Figs 1 and 2). We considered that these different responses of the GFP and lacZ reporters to Scd6-MS2-F tethering might arise from differences in codon optimality. However, the sTAI values for the GFP and lacZ coding sequences, 0.37 and 0.30, respectively, are both within one standard deviation of the mean sTAI value for all yeast genes of 0.35 . Given the negative correlation between sTAI values and change in mRNA abundance conferred by dhh1Δ for native mRNAs  (S11A Fig), it might be expected that lacZ mRNA (in the absence of tethered Scd6-MS2-F) would show increased abundance in dhh1Δ vs. WT cells; however we observed the opposite (S5F Fig), and we made qualitatively similar findings for a heterologous GAL1-lacZ mRNA (S5D Fig). Thus, these lacZ mRNAs behave more like mRNAs with optimized codons, except that the magnitudes of their reductions in dhh1Δ cells (2.5 to 4.5-fold) exceed the typical response of 10–20% reduced abundance seen for native codon-optimal mRNAs . In addition, one might expect that tethering Scd6-MS2 would evoke greater Dhh1-mediated reduction in mRNA abundance for the less codon-optimal lacZ versus more codon-optimal GFP mRNA , but again we found the opposite result. Hence, it is unlikely that differences in codon optimality underlie the different responses of these two reporters to tethered Scd6-MS2. Finally, it is noteworthy that most of the 53 mRNAs exhibiting the largest TE increases in scd6Δ cells do not exhibit increases in mRNA abundance (S7F Fig), indicating that Scd6 frequently decreases TE without reducing mRNA abundance of native mRNAs—as we observed for the lacZ reporter on tethering Scd6-MS2. On the other hand, a proportion of the 53 mRNAs do exhibit increased mRNA abundance in parallel with increased TE in scd6Δ cells (found in upper quartile of box plot in col. 1 of S7F Fig)—implying coupled repression of TE and mRNA abundance by Scd6—as we observed for the GFP reporter on tethering Scd6-MS2.
Another unexpected finding from the tethering assays was that reductions in reporter mRNA levels on tethering Scd6-MS-F are enhanced in dhh1Δ cells, increasing the repression ratio of GFP mRNA abundance (Fig 2C and 2D) and uncovering a repression of lacZ reporter mRNA abundance that was barely detectable in WT cells (Fig 5C and 5D). These observations might indicate that Dhh1 interferes with mRNA degradation evoked by tethered Scd6-MS2-F. This influence of Dhh1 was not seen for the five native mRNAs presented as exemplars of Scd6 translational repression (Fig 8C and S8A–S8D Fig), which all exhibit higher rather than lower mRNA levels in dhh1Δ cells. Moreover, increased mRNA levels in dhh1Δ cells holds for a large proportion of the group of 53 mRNAs whose TE was most strongly derepressed in the scd6Δ mutant (S7F Fig, col. 3). However, there is also a fraction of these mRNAs that do resemble the reporter mRNAs on Scd6-MS2 tethering in showing decreased mRNA abundance in dhh1Δ cells (S7F Fig, bottom quartile in col. 3). More work is required to understand the differing responses to Dhh1 for different Scd6 targets.
In summary, our results, in combination with previous findings on yeast Scd6 , support a model wherein recruitment of Scd6 to an mRNA, directed by or stabilized by its interaction with eIF4G, enables Scd6 to recruit other effectors of mRNA decapping/degradation and translational repression including, but not limited to, Dcp1/Dcp2 and Dhh1, and possibly also to interfere directly with recruitment of the 43S PIC by binding to the C-terminus of eIF4G (Fig 9, (i-iii)). Decapping by Dcp1/Dcp2 and subsequent degradation of the mRNA can proceed concurrently with translational repression (Fig 9, (iv)). Based on our findings that ccr4Δ enhances mRNA turnover and translational repression, we suggest that association of the Ccr4/Not complex with the mRNA, or deadenylation of the mRNA, interferes with the ability of Scd6 to associate with the mRNA or recruit decapping activators and translational repressors, thereby diminishing Scd6-enhanced mRNA degradation and translational repression in WT versus ccr4Δ cells. Further work will be required to determine whether Scd6 is recruited to specific mRNAs by 3’UTR sequences or RNA binding proteins unique to its mRNA targets, or whether intrinsic features of mRNAs (sequences or other binding proteins) confer a heightened sensitivity to Scd6 that would be recruited broadly to most mRNAs by eIF4G. Our identification of native mRNAs targeted by Scd6 for translational repression sets the stage for efforts to reconstitute the repressive function of Scd6 and its associated decapping activators in a purified translation system, and thereby elucidate their molecular mechanisms of translational control.
Materials and methods
Plasmids employed in this study are listed in Table 1. Plasmids containing constructs encoding FLAG-tagged MS2-CP fusions to Npl3, Sbp1, and Scd6 (S13 Fig, left) were constructed by first PCR-amplifying NPL3, SBP1 or SCD6 respectively with their native endogenous promoter (~500 bp upstream flanking sequence) plus their coding sequence minus the stop codon, from genomic DNA of WT strain BY4741, with primers containing a gene-specific restriction site at the N-terminus, and an XhoI site and XbaI/SpeI site at the C-terminus. The following primers were used: (i) NPL3 (forward primer with SpeI site, 5’-ACGAGGACTAGTTATCAATATGCAAATGCTCGGC-3’; reverse primer with XhoI and SpeI sites, 5’-ACGAGCACTAGTCTCGAGCCTGGTTGGTGATCTTTCACG); (ii) SBP1 (forward primer with XbaI site, 5’-ACGAGCTCTAGATCATCGAGCGGAAAATATTG-3’; reverse primer with XhoI and XbaI sites, 5’-ACGAGCTCTAGACTCGAGTTCTTGCTTTTCTTCAGAACC-3’); (iii) SCD6 (forward primer with SpeI site, 5’-ACGAGGACTAGTTGCTCGTAACAATCTTGG-3’; reverse primer with XhoI and SpeI sites, 5’-ACGAGGACTAGTCTCGAGAAATTCAACGTTGGAAGGAGG-3’). The amplified fragments were inserted between the XbaI/SpeI sites of YCplac111 to generate YCplac111-NPL3, YCpLac111-SBP1, or YCpLac111-SCD6, respectively. The MS2-CP CDS was PCR-amplified from plasmid pJC236 with primers containing an XhoI site and encoding a flexible linker (Gly-Gly-Gly-Gly-Gly-Ser) at the N-terminus, 3xFLAG epitopes, a stop codon and an overlapping sequence (for fusion PCR) at the C-terminus, using forward primer 5’-ATTCATCTCGAGGGTGGTGGTGGTGGTTCTGCTTCTAACTTTACTCAGTTCGTT-3’ and reverse primer 5’-TTACTTGTCATCGTCATCCTTGTAGTCGATGTCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTCGTAGATGCCGGAGTTTGCTGCGAT-3’. Next, the 3’UTR from each gene was amplified from genomic DNA of BY4741, with primers containing an upstream overlapping sequence (for fusion PCR) and a downstream XmaI site, using primers: (i) NPL3 3’UTR, forward primer 5’-CATGACATCGACTACAAGGATGACGATGACAAGTAAGCCATTTATATAGTTGAGAAAAAA-3’; reverse primer 5’-ATTTATCCCGGGTACCTATTCTGGCGTGTAATCCTTATCA-3’); (ii) SBP1 3’UTR, forward primer 5’-CATGACATCGACTACAAGGATGACGATGACAAGTAATTACTTCTTACCCACATCCCTATT-3’; reverse primer 5’-ATTTATCCCGGGTACCTCTCCGAGGTAGTGAACCATTGAG-3’); and (iii) SCD6 3’UTR, forward primer 5’-CATGACATCGACTACAAGGATGACGATGACAAGTAAAATGATGTTTCTATGTAAATTAAGTA-3’; reverse primer 5’-ATTTATCCCGGGTACCCTTTTCTTGTAGTTTGTTGTTCTTAC-3’). Fragments containing linker-MS2CP-FLAG-3’UTR sequences for each gene were generated by fusion PCR using the amplified fragments above, and inserted between the XhoI/XmaI sites of YCplac111-NPL3, YCplac111-SBP1, or YCplac111-SCD6, to generate the constructs pQZ125 (NPL3-MS2-F), pQZ126 (SBP1-MS-F) and pQZ127 (SCD6-MS2-F). Plasmids encoding MS2-FLAG control proteins pQZ128 (PNPL3-MS2-F), pQZ129 (PSBP1-MS2-F) and pQZ130 (PSCD6-MS2–F) (S13 Fig, right) were constructed by a strategy similar to that described above but with the NPL3, SBP1 and SCD6 CDSs absent from the final constructs and an ATG added at the beginning of the MS2 CP-encoding fragment. The specific primers for these constructions were: (i) PNPL3-MS2-F: forward primer for NPL3 promoter with SphI site 5’-ACGAGGGCATGCTATCAATATGCAAATGCTCGGCTC-3’; reverse primer for NPL3 promoter with ATG 5’-CATTATCCTTATGGTTTTAGCGTAATT-3’; forward primer for MS2-NPL3 3’UTR with ATG 5’-AATTACGCTAAAACCATAAGGATAATGGGTGGTGGTGGTGGTTCTGCTTCT-3’; reverse primer for MS2-NPL3 3’UTR with KasI site 5’-ATTTATGGCGCCTATTCTGGCGTGTAATCCTTATCA-3’); (ii) PSBP1-MS2-F: forward primer for SBP1 promoter with SphI site 5’-ACGAGGGCATGCTCATCGAGCGGAAAATATTGAAAA-3’; reverse primer for SBP1 promoter with ATG 5’-CATATTTTTCTTCGTTTGAGGGTTTTC-3’; forward primer for MS2-SBP1 3’UTR with ATG 5’-GAAAACCCTCAAACGAAGAAAAATATGGGTGGTGGTGGTGGTTCTGCTTCT-3’; reverse primer for MS2-SBP1 3’UTR with XmaI site 5’-ATTTATCCCGGGTACCTCTCCGAGGTAGTGAACCATTGAG-3’); (iii) PSCD6-MS2-F: forward primer for SCD6 promoter with SphI site 5’-ACGAGGGCATGCTGCTCGTAACAATCTTGGCCTAGC-3’; reverse primer for SCD6 promoter with ATG 5’-CATTGCCTTGCTGCTGTTTTTCGATGA-3’; forward primer for MS2-SCD6 3’UTR with ATG 5’-TCATCGAAAAACAGCAGCAAGGCAATGGGTGGTGGTGGTGGTTCTGCTTCT-3’; reverse primer for MS2-SCD6 3’UTR with XmaI site 5’-ATTTATCCCGGGTACCCTTTTCTTGTAGTTTGTTGTTCTTAC-3’). The GFP reporter plasmid pJC429 was described previously . The lacZ reporter plasmid pQZ131 was generated by PCR-amplifying the lacZ CDS sequence from GCN4-lacZ reporter plasmid p180, adding an SphI site and an ATG to the N-terminus (forward primer 5’-AAACTTGCATGCTTACGGAT-3’), and a PacI site to the C-terminus (reverse primer 5’-ACGAGCTTAATTAATTTTTGACACC-3’). The resulting fragment was inserted between the SphI/PacI sites of pJC429. Plasmid pQZ145 (DCP2 URA3) was generated by inserting into pRS316 a 4.3 kb XbaI-XmaI DCP2 fragment from pRS315-DCP2. Plasmids pQZ139 (ΔLSm-Scd6-MS2-F) and pQZ142 (ΔRGG-Scd6-MS2-F) were generated by deleting the CDS for amino acids Q3-D78 or S287-N318, respectively, of pQZ127 by site-directed mutagenesis (GenScript USA Inc). All plasmids were screened by restriction digestion and DNA sequencing was conducted to verify the presence of the intended inserts.
Yeast strains and growth conditions
Yeast strains employed in this work are listed in Table 2. Strain QZY126 was constructed by transforming HFY114 with a DNA fragment containing dhh1Δ::kanMX4 and including ~400 bp of sequences from both upstream and downstream of DHH1 that was PCR-amplified from genomic DNA of strain 3858 and selecting on YPD medium containing G418. Strain QZY128 was constructed by transforming strain CFY1016 (dcp2Δ::HIS3) harboring pQZ145 (DCP2 URA3) with the dhh1Δ::kanMX4 cassette as above, and evicting pQZ145 by counter-selection growth on 5-FOA medium. FZY843 was constructed by transforming CFY1016 with a fragment containing scd6Δ::KanMX4 that was PCR-amplified from yeast strain 5544. Strains SYY2352 and SYY2353 were constructed by transforming HFY114 with a DNA fragment harboring the scd6::KanMX6 null allele, which contains 400 bp from both upstream and downstream of SCD6 with the coding region replaced by a 1447-bp KanMX6 cassette. Gene disruptions were confirmed by PCR analysis of chromosomal DNA using the appropriate primers. Unless otherwise noted, all strains were grown at 30°C in synthetic complete medium without leucine or uracil (SC-L-U) with 2% galactose/2% raffinose replacing dextrose. All cultures were grown for at least two cell divisions and harvested at mid-log phase (OD600 = 0.6–0.7).
Biochemical analyses using yeast cell extracts
For Western blot analysis, whole-cell extracts (WCEs) from at least three biological replicates (independent transformants) were prepared by trichloroacetic acid (TCA) extraction as previously described . Aliquots of WCEs were resolved by 4–20% SDS-PAGE, transferred to PVDF membrane and probed with antibodies against GFP (Covance), Prt1 , FLAG epitope (Sigma), or Gcd6 . Immune complexes were detected using the Pierce enhanced chemiluminescence (ECL) system and autoradiography; and signal intensities were quantified by scanning densitometry using NIH ImageJ software. Assays of β-galactosidase activity in WCEs were performed as described previously . At least four biological replicates (and two technical replicates per transformant) were employed for all β-galactosidase assays.
Polysome analysis and RNA extraction from sucrose gradients
For polysomes profiles, 300 mL of cells were treated with 50 μg/ml cycloheximide for 5 min prior to harvesting. WCEs were prepared in 1x breaking buffer (20 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 mM MgCl2, 1 mM DTT, 50 μg/ml cycloheximide, 1 mM PMSF, Complete EDTA-free Protease Inhibitors, 1U/μl SUPERase-In RNase inhibitor) by vortexing with glass beads, followed by two cycles of centrifugation for 10 min at 15,000 rpm at 4°C. 15 OD260 units of cleared lysate were loaded on 15%-45% (w/w) sucrose gradients prepared on a Biocomp Gradient Master in 1x breaking buffer and centrifuged at 41,000 rpm for 73 min at 4°C in a Beckman SW41Ti rotor. Gradients were fractionated with a Brandel Fractionation System and ribosomal peaks were visualized at 254 nm with an Isco UV detector. Fractions (0.7 mL) were precipitated overnight at -20°C using 1.5 volumes RNA precipitation mix (95% EtOH, 5% NaOAc), and centrifuged at 15,000 rpm for 30 min to pellet RNA/protein. Pellets were washed in 1 mL cold 80% EtOH, dried in a speed vacuum, and resuspended in 100 μl AE buffer (50 mM NaOAc, 10 mM EDTA, pH 5.2) with 1% SDS. Fractions were extracted by adding 350 μl QIAzol lysis reagent and incubating 5 min at room temperature, adding 70 μL chloroform and incubating 2–3 min at room temperature, and centrifuging at 15,000 rpm for 15 min at 4°C. The aqueous phase (~300 μl) was transferred to a new collection tube, thoroughly mixed with 1 volume of 100% EtOH, and applied to a purification column (RNA Clean and Concentrator™ kit, Zymo Research) to isolate RNA following the vendor’s instructions. RNA from each polysomal fraction was eluted with 25 μl RNase-free water, and 2 μl/reaction were used for first strand cDNA synthesis, as described below.
Total RNA isolation, RT-qPCR and Northern blot analysis
Yeast cultures (25 mL) were harvested by centrifugation, and the resulting pellets were resuspended in 400 μL AE buffer (50 mM NaOAc, 10 mM EDTA, pH 5.2) with 1% SDS. Cell suspensions were extracted twice with AE buffer-equilibrated phenol, twice with phenol/chloroform/isoamyl alcohol (25:24:1), once with chloroform/isoamyl alcohol (24:1), and then precipitated at -20°C with 2 volumes RNA precipitation mix (95% EtOH, 5% NaOAc). RNA pellets were recovered by centrifugation at 15,000 rpm for 30 min (4°C), washed once with 1 mL cold 80% EtOH, dried in a speed vacuum, and resuspended in 50 μL RNase-free water. Total RNA concentration was calculated at A260 using a NanoDrop spectrophotometer. For RT-qPCR, reverse transcription was carried out using SuperScript III First-Strand cDNA Synthesis SuperMix (Invitrogen), with random primers and either 1 μg total cellular RNA from above or 2 μL polysomal RNA isolated from sucrose gradient fractions. qPCR was carried out using Brilliant III Ultra-Fast SYBR Green Master Mix (Agilent) in a Mx3000P System (Stratagene) and the following oligonucleotide pairs: GFP (5’-GGCTAGCAAAGGAGAAGAACTC-3’; 5’-CCGTATGTTGCATCACCTTC-3’), lacZ (5’-ACCAACGTAACCTATCCCATTAC-3’; 5’-TTCCTGTAGCCAGCTTTCATC-3’), ACT1 (5’-TGTGTAAAGCCGGTTTTGCC-3’; 5’-GATACCTCTCTTGGATTGAGCTTC-3’). Levels of reporter mRNA relative to actin were calculated using the ΔCt method. For Northern blot analysis, 10 μg of total RNA/lane were denatured with glyoxal/DMSO, separated on 1.4% agarose gels, transferred to nylon membranes, and probed with [32P]-end-labeled DNA oligonucleotides complementary to GFP or yeast PYK1 transcripts. Blots were exposed to PhosphorImager screens and scanned using a Storm scanner.
Measurements of GFP mRNA half-lives. A previously described protocol  was employed with the following modifications. Yeast transformants were cultured in 330 mL of SC-U,-L with 2% Galacose and 2% Raffinose to an A600 of ~0.8. An aliquot of 30 mL, representing the “0 min” time point, was poured onto ice and collected by centrifugation at 3,000 rpm in a Beckman JS-4.2 rotor for 5 min at 4°C. The cell pellet was resuspended and transferred to a 1.5 mL Eppendorf tube, and spun at 4°C in a microfuge at top speed (15,000 rpm) for 2 min. Residual medium was aspirated and cell pellets were immediately frozen on dry ice. The rest of the culture was collected by centrigugation at room temperature for 8 min at 7,000 rpm in an SLA3000 rotor. The cell pellet was resuspended in an equal volume of SC-U,-L 4% glucose medium pre-warmed to 30°C, and aliquots of 30 mL were harvested exactly as described for the “0 min” sample at 2-, 4-, 6-, 8-, 10-, 15-, -20, -30, -40, and 60 min after glucose containing medium was added. Frozen cells pellets were stored at -80°C until being thawed for isolation of total RNA and qRT-PCR analysis, which was conducted as described above.
Statistical analysis of tethering data
Changes in reporter protein expression (ΔGFP protein or Δβ-galactosidase) or reporter mRNA abundance (ΔGFP mRNA or ΔlacZ mRNA) were calculated as ratios of mean values of protein or mRNA expression measured in 3 or more biological replicates of transformants expressing Scd6-MS2-F vs. MS2-F alone. The propagated S.E.M.s for the resulting mean ratios were computed as (X/Y)*(√[(SEx/x)2+(SEy/y)2]), where X, SEx, and x are the mean, standard error of the mean, and highest values for reporter protein input, respectively; and Y, SEy, and y are the corresponding values for the mRNA input. Unpaired t-tests were performed to compare the mean changes in reporter protein or mRNA expression between wild type and mutant strains. Changes in TE of reporter mRNA on tethering Scd6-MS2 were determined by calculating the TE of the reporter, as the ratio of reporter protein to reporter mRNA expression, measured in 3 or more pairs of independent transformants expressing Scd6-MS2-F or MS2-F alone, from which the mean ΔTE and S.E.M. was calculated. Unpaired t-tests were performed to compare the resulting mean ΔTE values between wild type and mutant strains.
Ribosome footprint profiling and RNA-Seq
Ribosome profiling and RNA-Seq analysis were conducted in parallel essentially as described previously  on isogenic strains in the W303 background HFY114 (WT), CFY1016 (dcp2Δ), SYY2353 (scd6Δ), QZY126 (dhh1Δ), FZY843 (dcp2Δscd6Δ), and QZY128 (dcp2Δdhh1Δ), providing two biological replicates of each genotype. Strains growing exponentially in YPD medium at 30°C were harvested by vacuum filtration at room temperature, and quick-frozen in liquid nitrogen. Cells were lysed in a freezer mill with lysis buffer (20 mM Tris [pH 8.0], 140 mM KCl, 1.5 mM MgCl2, 1% Triton, 500 μg/mL cycloheximide). For ribosome footprint library preparation, approximately 60 A260 units of extract were treated with RNAse I at 15 U per OD260 unit (Ambion, #AM2295) for 1h at 25°C on a Thermomixer at 700 rpm, and 80S ribosomes were purified by sedimentation through a sucrose density gradient as described . Ribosome-protected mRNA fragments (footprints) were purified by phenol and chloroform extractions . Following size selection and dephosphorylation, a Universal miRNA cloning linker (Synthesized by Integrated DNA Technologies) was ligated to the 3’ ends of footprints, followed by reverse transcription, circular ligation, rRNA subtraction, PCR amplification of the cDNA library, and DNA sequencing with an Illumina HiSeq system. For RNA-Seq library preparation, total RNA was purified using miRNeasy Mini kit (Qiagen) from aliquots of the same extracts (30 OD260 units) used for footprint library preparation, 5 μg total RNA was randomly fragmented at 70°C for 12 min in fragmentation reagent (Ambion #AM8740). Fragment size selection, library generation and sequencing were carried out as above, except Ribo-Zero Gold rRNA Removal Kit (Yeast) was employed to remove rRNAs after linker-ligation in lieu of poly(A) selection. As described , linker sequences were trimmed from Illumina reads and the trimmed fasta sequences were aligned to the S. cerevisiae ribosomal database using Bowtie . The non-rRNA reads (unaligned reads) were then mapped to the S. cerevisiae genome using TopHat . Wiggle track normalization for viewing RPF or RNA reads in the IGV browser was conducted as follows. Wiggle files were produced from the alignment file, one each for genes on the Watson or Crick strand. The total reads on both strands were summed and a normalization factor q was calculated as 1,000,000,000/(total reads on W+C strands). Wiggle files were then regenerated by multiplying all reads by the factor q, yielding the number of reads per 1,000 million total reads (rpkm). Statistical analysis of changes in mRNA, RPFs, or TE values between two replicates each of any two strains being compared was conducted using DESeq2 , excluding any genes with less than 10 total mRNA reads in the 4 samples combined.
RNA-Seq analysis of strains SYY2352 (scd6Δ), SYY2353 (scd6Δ), SYY2686 (dhh1Δ), SYY2674 (pat1Δ), and SYY2680 (lsm1Δ) was conducted as described previously  after culturing cells in YPD at 30°C; and the results will be described in full in a future publication.
For all notched box-plots, constructed using a web-based tool at http://shiny.chemgrid.org/boxplotr/, the upper and lower boxes contain the 2nd and 3rd quartiles and the band gives the median. If the notches in two plots do not overlap, there is roughly 95% confidence that their medians are different.
Accession numbers of deposited data
RNA-Seq data employed in the analysis of mRNA changes in scd6Δ, dhh1Δ, pat1Δ, and lsm1Δ strains for the group of 83 mRNAs derepressed in scd6Δ cells have been deposited in the NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) under the accessions numbers GEO:GSE107841 and GEO:GSE114428. All other RNA-Seq or Ribo-Seq data generated in this study are deposited separately in GEO:GSE114892.
S1 Fig. Tethered Scd6-MS2-F and Dhh1-MS2 confer similar decreases in the half-life of GFP reporter mRNA.
(A) Transformants from Fig 1B–1C expressing MS2-F, Scd6-MS-F, Dhh1-MS2, and WT strain BY4741 harboring empty vector YCpLac111 (Vector), all harboring GFP reporter plasmid pJC429, were analyzed for GFP protein expression as in Fig 1B. (B-C) Transformants of WT strain HFY114 containing expression plasmids for Scd6-MS2-F (pQZ127) or MS2-F (pQZ130) (B), or Dhh1-MS2 (pJC236) or MS2-F (pQZ130) (C), and GFP reporter plasmid pJC429, were cultured in SC-L-U with 2% galactose/2% raffinose and shifted to SC-L-U with 2% glucose to repress reporter mRNA transcription. Total RNA was isolated from cells harvested at the indicated times and subjected to qRT-PCR to measure the amount of GFP mRNA remaining at each time point relative to ACT1 mRNA. The t1/2 values were calculated from the slopes of the best-fit lines shown in the plots, k, for the initial rates of decay, using the equation t½ = 0.693/k. Data from two biological replicates are shown for each construct, with the results of an unpaired Student’s t-test on the mean t1/2 values measured for Scd6-MS2-F (B) or Dhh1-MS2 (C) vs. MS2-F alone indicated: *, P < 0.05.
S2 Fig. Tethering Npl3-MS2-F or Sbp1-MS2-F does not reduce GFP reporter protein expression in vivo.
(A-B) WT cells (BY4741) transformed with plasmids expressing MS2NPL3-F (pQZ128) or Npl3-MS2-F (pQZ125) and GFP reporter plasmid pJC429 were analyzed for GFP protein expression as in Fig 1B and 1C. Average results (±S.E.M.s) from at least three biological replicates are represented. (C-D) WT cells (BY4741) were co-transformed with plasmids encoding MS2SBP1-F (pQZ129) or Sbp1-MS2-F (pQZ126) and pJC429 were analyzed for GFP protein expression as in Fig 1B and 1C. Mean values (± S.E.M.s) were determined from at least three biological replicates. (E) WCEs of WT cells transformed with plasmids expressing the indicated MS2 fusion proteins were subjected to Western blot analysis using antibodies against FLAG (upper) or Prt1 (lower).
S3 Fig. Control experiment showing that tethering MS2-F does not reduce GFP reporter protein expression in dcp2Δ cells.
Transformants of dcp2Δ strain CFY1016 harboring expression plasmids for MS2-F (pQZ130) or empty vector YCplac111 and GFP reporter pJC429, were analyzed for GFP protein expression as in Fig 1B–1D.
S4 Fig. Polysome size distribution of GFP reporter mRNA is altered on tethering Scd6-MS2-F.
(A-B) Results from three biological replicate gradients of dcp2Δ transformants harboring the GFP reporter and expressing MS2-F (A) or Scd6-MS2-F (B), which were averaged to produce the results shown in Fig 3B. WCEs were separated by velocity sedimentation on sucrose density gradients and fractionated with continuous monitoring at A254. The abundance of GFP mRNA was quantitated by RT-qPCR in total RNA extracted from the gradient fractions and plotted as the percentage of total signal in the gradient.
S5 Fig. Additional tethering experiments and controls for the lacZ reporter.
(A) Repression of the lacZ reporter by tethered Scd6-MS2-F is independent of native Scd6. Transformants of scd6Δ strain 5544 expressing the MS2-F or Scd6-MS2-F fusions from Fig 1 and containing the lacZ reporter on pQZ131 were analyzed for β-galactosidase as in Fig 5B. (B) Expressing Scd6-MS2-F does not affect expression of heterologous GCN4-lacZ or GAL1-lacZ reporters lacking MS2 CP binding sites. β-galactosidase activities were determined in WCEs from WT (BY4741) cells harboring plasmids containing a GCN4-lacZ reporter (p180) or GAL1-lacZ reporter (pCGS286) and expressing either MS2-F (pQZ130) or Scd6-MS2-F (pQZ127), cultured in synthetic complete medium without leucine or uracil (SC-L-U) containing 2% dextrose as carbon source, for p180, or 2% galactose/2% raffinose for pCGS286. (C) Tethering Npl3-MS2-F or Sbp1-MS2-F does not affect expression of the MS2 CP lacZ reporter. WCEs from WT cells (BY4741) containing either empty vector or the indicated MS2 fusion protein, and pQZ131, were analyzed for β-galactosidase expression as in Fig 5B. (D) Expression of a heterologous GAL1-lacZ reporter lacking MS2CP binding sites is reduced in dhh1Δ cells. β-galactosidase activities were measured in WCEs of isogenic WT (BY4741) or dhh1Δ (3858) strains containing a GAL1-lacZ reporter on pCGS286, cultured as in Fig 5B. (E-G) Expression of the lacZ reporter is altered in dhh1Δ and dcp2Δ cells independently of tethered Scd6-MS2-F or MS-F. Transformants of WT (BY4741) or dhh1Δ (3858) strains containing empty vector or the expression plasmids for MS2-F or Scd6-MS2-F described in Fig 1, and pQZ131, were analyzed for expression of β-galactosidase (E) and lacZ mRNA (F) as in Fig 5B and 5C. (G) Transformants of dcp2Δ strain CFY1016 containing the MS2-F expression plasmid or empty vector and pQZ131 (3858) were analyzed for expression of lacZ mRNA. Mean values (± S.E.M.s) were determined from at least three biological replicates. Determination of P-values from significance testing of differences in mean values using an unpaired Student’s t-test, were conducted as described in Supplementary file “Data Analysis and Explanation of Source Files”. P-values are summarized as: **, P <0.01; *, P <0.05.
S6 Fig. High reproducibility of RNA-Seq and Ribo-Seq data in biological replicates.
(A-L) Scatterplots of RNA (A, C, E, G, I, K) or ribosome footprints (B, D, F, H, J, L) read densities (number of reads mapping to each gene’s CDS normalized by the CDS length) for all expressed genes for biological replicates of the following strains: (A-B) HFY114 (WT); (C-D) SYY2353 (scd6Δ); (E-F) QZY126 (dhh1Δ); (G-H) FZY843 (dcp2Δscd6Δ); (I-J) QZY128 (dcp2Δdhh1Δ); (K-L) CFY1016 (dcp2Δ). Pearson correlation coefficients (r) are indicated in each plot.
S7 Fig. Scd6 and Dhh1 cooperate in repressing mRNA abundance and translational efficiencies of a subset of native mRNAs.
(A) Notched box-plots of log2(ΔmRNA) values in the indicated mutants for 346 mRNAs exhibiting ≥2.0-fold increased mRNA abundance in dhh1Δ(z) versus WT cells (at FDR<0.01). TE changes were calculated from the data sets described in Fig 7C. (B-C) Hierachical clustering analysis conducted as in Fig 8A and 8B RNA-Seq data from scd6Δ (SYY2353), dhh1Δ (QZY126, dhh1Δ(z)), and WT (HFY114) strains. Approximately 50 genes were removed for which no data were available in one of the strains, or where the log2(ΔmRNA) value was >4 or <-4 in one of the mutant vs. WT comparisons, after which separate clustering analysis was performed on two sets of mRNAs in which all log2(ΔmRNA) values fell between -2 or +2 (panel B, 5213 mRNAs), or in which the log2(ΔmRNA) value for one of the mutants was < -2 or > +2 (panel C, 124 mRNAs). The color key for log2(ΔmRNA) values is indicated above each analysis. (D-E) Notched box-plots of log2(ΔTE) values in the indicated mutants for the 83 mRNAs analyzed in Fig 7A exhibiting ≥1.4-fold increased mRNA abundance in scd6Δ versus WT cells (D); and for the same 346 mRNAs analyzed in (A), exhibiting ≥2.0-fold increased mRNA abundance in dhh1Δ(z) versus WT cells (E). TE changes were calculated from the data sets described in Fig 7C. (In panels A, C, E & F, the double mutants carry an (*) to indicate that their values have been compared to the dcp2Δ single mutant rather than to WT.) (F) Notched box-plots of log2(ΔmRNA) values in the indicated mutants for 53 mRNAs exhibiting ≥1.33-fold increased TE in scd6Δ versus WT cells described in Fig 7E. The RNA changes were calculated from the indicated data sets described in (A). For panels A, D, E, & F, the changes in mRNA abundance or TE for the relevant group of mRNAs found in each of the indicated mutants were plotted irrespective of whether the changes exhibit statistical significance in that mutant, to allow a coherent comparison of the behavior of the complete cohort of mRNAs across the entire panel of mutants. Statistical significance is evaluated for differences in the median changes found in the different mutants, with non-overlapping notches indicating with 95% confidence that the median changes found for two mutants differ from one another.
S8 Fig. Exemplar genes exhibiting increased TE in both scd6Δ and dhh1Δ versus WT cells.
(A-D) Integrated Genomics Viewer (Broad Institute) displays of ribosome-protected fragments (RPFs) and mRNA reads across the indicated genes from two biological replicates each for WT, scd6Δ and dhh1Δ strains, shown in units of rpkm. Position of the CDS (magenta) is shown at the bottom with the scale in bp; scales of rpkm for each track are on the left, and calculated ΔRPF, ΔmRNA and ΔTE values between each mutant and WT are on the right.
S9 Fig. Scd6 and Dhh1 repress mRNA abundance of genes involved in metabolism of energy reserves and other carbohydrates.
GO term analysis conducted using web tool Funspec at http://funspec.med.utoronto.ca/ applying Bonferroni correction. The functional categories showing enrichment derive from the MIPS database. k/n/f: number of genes in MIPS category/number of genes in up-regulated list/total number of genes in MIPS category. (A) 346 mRNAs exhibiting ≥2.0-fold increased mRNA abundance in dhh1Δ(z) versus WT cells at FDR<0.01; analyzed in S7A Fig. (B) 83 mRNAs exhibiting ≥1.4-fold increased mRNA abundance in scd6Δ versus WT cells at FDR<0.01; analyzed in Fig 7A.
S10 Fig. Dhh1 occupancy tends to be elevated for mRNAs showing increased abundance, ribosome occupancy or TE in dhh1Δ vs. WT cells.
(A-C) Boxplot analysis of changes in ribosome occupancy (A), TE (B) or mRNA abundance (C) versus Dhh1 RIP-seq enrichment values from Miller et al (2018). The latter were equally divided into five pentiles of 739 genes from lowest to highest enrichment values and plotted against the log2(Δribo) values (A), log2(ΔTE) values (B), or log2(ΔmRNA) values determined by ribosome profiling analysis of dhh1Δ strain (QZY126, dhh1Δ(z)) and WT strain HFY114. The Pearson correlation coefficients for the relationship between log2(Δribo) values (panel A), log2(ΔTE) values (panel B), or log2(ΔmRNA) values (panel C) and Dhh1 enrichment for all mRNAs are 0.31 (P = 3 X 10−81), 0.18 (P = 1 X 10−29), and 0.27 (P = 6 X 10−62), respectively.
S11 Fig. Low codon optimality is associated with increased mRNA abundance in dhh1Δ vs. WT cells.
(A-C). Yeast mRNAs were binned by sTAI values, a measure of overall codon optimality  and the log2(ΔmRNA) values measured by RNA-Seq in three independent analyses of dhh1Δ vs. WT strains (described in S7A Fig) were displayed in a box-plot for each bin. In all cases, the bin containing the lowest sTAI optimality scores (median of ~0.25) shows greater increases in mRNA expression in the dhh1Δ mutant vs WT compared to the bin containing the highest sTAI optimality scores (median of ~0.61), as observed previously . However, the magnitude of this difference is relatively less for the dhh1Δ datasets from Jungfleisch et al.  (B) and the current study (C) compared to that of Radhakrishnan et al.  (A).
S12 Fig. Low codon optimality appears to play a minor role in conferring repression of mRNA abundance by Scd6.
(A) Yeast mRNAs were binned by sTAI values as in S11 Fig and the log2(ΔmRNA) values measured by RNA-Seq analysis of scd6Δ (SYY2353) vs. WT (HFY114) cells. The bin containing the lowest sTAI optimality scores (median of ~0.25) shows greater increases in mRNA expression in the scd6Δ mutant vs WT compared to the bin containing the highest sTAI optimality scores (median of ~0.61), as observed previously for a dhh1Δ mutant . (B) The sTAI values are significantly higher for the group of 83 mRNAs found by RNA-Seq to exhibit elevated abundance in scd6Δ strains (SYY2352 and SYY2353) vs. WT strain (HFY114), compared to all mRNAs.
S13 Fig. Schematics of expression constructs for MS2 fusion and control proteins for in vivo tethering assays.
Scd6-MS2-F, Npl3-MS2-F, and Spb1-MS-F fusion proteins were expressed under the control of their native promoters and 5’UTR and 3’UTR sequences, with the complete CDS of each protein fused in-frame at the C-terminus to a 5-amino acid linker, followed by the coding sequences for MS2 CP and three FLAG epitopes. The MS2-FLAG control constructs are identical except that they lack the respective Scd6/Npl3/Spb1 CDSs. The fusion protein expression constructs are contained on the following plasmids: ΡSCD6-SCD6-MS2-F (pQZ127), ΡNPL3-NPL3-MS2-F (pQZ125), and ΡSBP1-SBP1-MS2-F (pQZ126). The corresponding MS2-F control constructs are as follows: ΡSCD6-MS2-F (pQZ130), ΡNPL3-MS2-F (pQZ128), and ΡSBP1-MS2-F (pQZ129).
S1 Text. Analysis and Explanation of Supporting Data Files.
Description of procedures employed to analyze the source data found in supporting data files S1-S10 to (i) calculate mean values from replicate determinations of reporter protein or reporter mRNA expression, reporter TE, or changes in these parameters between mutant and WT cells, and conduct statistical analysis of observed differences in the corresponding means, for results in Figs 1, 2, 4, 5, 6, S2, S3 and S5; (ii) conduct statistical analysis of differences in observed mean values of reporter mRNA levels in different polysome gradient fractions for results in Fig 3; (iii) calculate reporter mRNA mean half-lives and conduct statistical analysis of differences in the corresponding mean values for results in S1 Fig.
S1 Data. Source data and statistical analysis for Fig 1C, 1D and 1F.
For Fig 1B and 1C, GFP protein expression was analyzed by quantification of the GFP protein and loading control (LC, Prt1) signals on immunoblots by densitometry and the GFP/LC ratio was calculated for each biological replicate (BR) of Scd6-MS2-F or MS2-F expressing transformants. The mean GFP/LC ratio, and both the standard deviation and S.E.M., were calculated from between 3 and 6 biological replicates of Scd6-MS2-F or MS2-F expressing transformants. An unpaired Student’s t-test was performed comparing the mean GFP/LC ratios between the Scd6-MS2-F and MS2-F transformants and the magnitude of the P-values are summarized as: **, P <0.01; *, P <0.05 in Fig 1C. For Fig 1D, GFP mRNA expression was analyzed by determining the mean 2-Ct values from 3 technical replicates (TR) for GFP mRNA, and also for ACT1 mRNA for the same RNA samples, and the ratio of GFP/ACT1 2-Ct values was calculated from the ratio of the resulting mean 2-Ct values. The GFP/ACT1 ratios thus determined from five or more biological replicates were averaged for Scd6-MS2-F or MS2-F expressing transformants, and the mean values and both the standard deviation and S.E.M. were calculated. An unpaired Student’s t-test was performed comparing the mean GFP/ACT1 2-Ct values between the Scd6-MS2-F and MS2-F transformants and the magnitudes of the P-values are summarized as: **, P <0.01; *, P <0.05 in Fig 1D. For Fig 1F, changes in GFP protein or mRNA expression, or in TE values, on tethering Scd6-MS2 vs. MS2 alone were calculated as follows. The change in GFP protein (ΔGFP Protein) was calculated as the ratio of the mean values from panel C for Scd6-MS2-F vs. MS2-F. The propagated S.E.M. for the resulting ratio of means was calculated using the formula: (X/Y)*(√[(SE_x/x)2+(SE_y/y)2]), where X, SE_x, and x are the mean, standard error of the mean, and highest values for Scd6-MS2-F, respectively; and Y, SE_y, and y are the corresponding values for MS2-F. The change in GFP mRNA (ΔGFP mRNA) and S.E.M. were calculated in the same way, and the resulting mean and S.E.M.s for ΔGFP protein and ΔGFP mRNA were entered in Fig 1F. Changes in TE of GFP mRNA on tethering Scd6-MS2 vs. MS2 alone were calculated as follows, noting that aliquots of cell cultures used for GFP protein and GFP mRNA were taken from the same biological replicate culture. For each pair of biological replicates expressing Scd6-MS2-F or MS2-F, the change in GFP protein and change in GFP mRNA were calculated as the Scd6-MS2-F/MS2-F ratios from the corresponding GFP protein and GFP mRNA values (normalized to Prt1 or ACT1 mRNA as described above), for that pair of transformants. The ratio of the resulting changes in GFP Protein to changes in GFP mRNA was calculated to determine the change in TE (ΔTE) on tethering Scd6-MS2-F vs. MS2-F for that pair of transformants. The mean ΔTE and S.E.M. was calculated by averaging the ΔTE values for six different pairs of biological replicates of Scd6-MS2-F and MS2-F transformants, and entered in Fig 1F.
For Fig 2B and 2C, GFP protein (B) and GFP mRNA (C) expression data in each WT and mutant strain were analyzed exactly as described for Fig 1C and 1D, respectively, in S1 Data for the WT strain; and an unpaired Student’s t-test was performed comparing the mean GFP/LC ratios (B) mean GFP/ACT1 2-Ct values (C) between the Scd6-MS2-F and MS2-F transformants in each strain. For Fig 2D, changes in GFP protein or GFP mRNA expression on tethering Scd6-MS2 vs. MS2 alone in each strain were calculated as described for Fig 1F in supporting file S1 Data for the WT strain. An unpaired Student’s t-test was performed comparing the mean ΔGFP protein or ΔGFP mRNA values between different strains using the propagated S.E.M. values (calculated using the formula employed for Fig 1F) and the number (N) of BRs examined in each strain in comparing Scd6-MS2-F to MS2-F transformants. For Fig 2E, changes in TE of GFP mRNA on tethering Scd6-MS2 vs. MS2 alone in each strain were calculated as described for Fig 1F in supporting file S1 Data for the WT strain. An unpaired Student’s t-test was performed comparing the mean ΔTE values between pairs of Scd6-MS2-F and MS2-F transformants determined in each mutant vs. WT. Magnitudes of P-values from t-tests are summarized in Fig 2B–2E as: **, P <0.01; *, P <0.05; n.s., not significant.
S3 Data. Source data and statistical analysis for Fig 3B and 3C.
For each of three biological replicate transformants (BR1-BR3) expressing Scd6-MS2-F or MS2-F alone, 2-Ct values were determined in triplicate (technical replicates TR1-TR3) for GFP or ACT1 mRNA from the RNA isolated from each gradient fraction and averaged. The average 2-Ct value for each fraction was plotted as a proportion of the sum of the average 2-Ct values for all gradient fractions in Fig 3B for GFP mRNA and in Fig 3C for ACT1 mRNA. An unpaired Student’s t-test was performed comparing the mean proportions of GFP mRNA in each fraction for Scd6-MS2-F versus MS2-F transformants, and the magnitudes of P-values are summarized in Fig 3B and 3C as: **, P <0.01; *, P <0.05.
(A-B, D) GFP protein (A) or GFP mRNA (B,D) expression data in WT and ccr4Δ (A-B) or caf1Δ (D) strains were analyzed exactly as described for Fig 1C and 1D in supporting file S1 Data, respectively, for the WT strain. (C-D) Changes in GFP protein (C) or GFP mRNA expression (C-D) on tethering Scd6-MS2 vs. MS2 alone in each mutant or WT strain were analyzed as described for Fig 2D in supporting file S2 Data. Changes in TE of GFP mRNA on tethering Scd6-MS2 vs. MS2 alone in each mutant or WT strain (C) were analyzed as described for Fig 2E in supporting file S2 Data.
For Fig 5B, units of β-galactosidase activity measured for three or more biological replicates (BR) of Scd6-MS2-F- or MS2-F-expressing transformants of each WT or mutant strain were averaged; and an unpaired Student’s t-test was performed comparing the mean activities between the Scd6-MS2-F and MS2-F transformants in that strain. For Fig 5C, lacZ mRNA expression data in each strain were analyzed exactly as described for GFP mRNA for Fig 1D in supporting file S1 Data, for the WT strain. For Fig 5D, changes in β-galactosidase activity or lacZ mRNA expression on tethering Scd6-MS2 vs. MS2 alone in each strain were analyzed as described for Fig 2D in supporting file S2 Data. For Fig 5E, changes in TE of lacZ mRNA on tethering Scd6-MS2 vs. MS2 alone in each strain were analyzed as described for Fig 2E in Source Data File 2.
GFP protein (Fig 6D & 6F), GFP mRNA (Fig 6E), and β-galactosidase (Fig 6G) expression data in WT transformants expressing WT or mutant derivatives of Scd6-MS2-F, or MS2-F alone, were analyzed as described for Fig 1C and Fig 1D in supporting file S1 Data, and for Fig 5B in file S5 Data, respectively.
S7 Data. Source data and statistical analysis for S1B and S1C Fig.
Analysis of RT-qPCR data to calculate the percent normalized GFP mRNA found at time = 0 min remaining at each time point following a shift from galactose to glucose as carbon source; determination of the slopes (k) of semi-log plots of the resulting values versus time; and calculation of t1/2 values as 0.693/k were analyzed for WT strains expressing the GFP reporter and Scd6-MS2-F or MS2-F (B); and Dhh1-MS2 or MS2-F (C).
GFP protein expression data in WT transformants expressing Npl3-MS2-F or MS2-F alone, both expressed from the NPL3 promoter (S2B Fig); or Sbp1-MS2-F or MS2-F alone, both expressed from the SBP1 promoter (S2D Fig), were analyzed as described for Fig 1C in supporting file S1 Data.
S10 Data. Source data and statistical analysis for S5A–S5G Fig.
For S5A–S5E Fig, β-galactosidase expression data were analyzed as described for Fig 5B in supporting file S5 Data. For S5F and S5G Fig, lacZ mRNA expression data were analyzed as described for Fig 5C in file S5 Data.
We thank Jeff Coller for generous gifts of strains and plasmids and Gavin Hanson for assistance on calculating sTAI values. We thank members of our laboratories and those of Tom Dever and Jon Lorsch for many helpful comments.
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