Figure 1.
The rrp6l1-3 and rrp6l2-3 mutants affect flowering time and gene expression.
(A) The late-flowering phenotype of rrp6l1 and rrp6l2 mutants grown under long day conditions. Flowering time was measured as rosette leaf number at bolting. To control for effects of ecotype, the phenotype of mutants was compared to wild-type plants of Col-0 and Ws ecotypes. Error bars represent standard deviation (SD). (B) The late-flowering phenotype of rrp6l1-3 rrp6l2-3 mutants grown under short day conditions. 66-day-old plants are shown. (C) Effect of rrp6l1-3 and rrp6l2-3 mutations on the expression of the FLC, SOC1 and FT. RT-PCR showed that the rrp6l1-3 rrp6l2-3 double mutant (rrp6l1/2), has increased expression of FLC and decreased expression of SOC1 and FT, which act downstream of FLC. (-RT) is the no reverse transcriptase control. (D) The expression of FCA, FPA, FLD, FVE, FLM, and LDL2, genes involved in regulation of flowering time in the autonomous flowering pathway, is not affected in rrp6l1/2 mutants. (E) FLC expression is not affected in AtCSL4-2 T-DNA mutant and RRP41 iRNAi line. RRP41 corresponds to the iRNAi line grown without estradiol, and rrp41-i corresponds to line grown on estradiol-containing medium, to induce the RNAi-mediated knockdown of RRP41. TUBULIN 2 and ACTIN 7 were used as loading controls.
Figure 2.
The effect of the rrp6l1-3 and rrp6l2-3 mutations on expression of FLC sense and antisense transcripts, and on H3K4 methylation.
(A) The diagram of the FLC gene based on analysis of the transcription unit [38] Vertical bars and numbers denote the exons of the FLC sense transcript. The transcription start site of the FLC sense mRNA is indicated by an arrow. Two antisense transcripts, AS I (proximal) and AS II (distal), are depicted below the FLC diagram. Antisense transcripts are alternatively polyadenylated, with a proximal poly(A) site in sense intron 6 and a distal poly(A) site in the sense promoter region. Grey boxes correspond to AS I and II exons, and grey lines correspond to the spliced regions of the antisense RNAs. Horizontal bars (letters a, A to G and 3′-untranslated region, 3′UTR), correspond to the FLC regions used in qRT-PCR and ChIP. Arrowheads correspond to position of primers used for RT-PCR amplification of AS I and II. Blue bars correspond FCA binding region, yellow bar correspond to the FPA binding region. (B) Nascent FLC expression significantly increased in rrp6l1/2 mutants. Expression of FLC was measured by qRT-PCR and is shown relative to FLC expression in Col-0 and Ws wild type plants; (C) Expression of AS I and AS II transcripts in rrp6l1/2 mutants. Expression of AS I and AS II in rrp6l1/2 was compared to their expression in Col-0 wild type plants by qRT-PCR. The antisense transcript levels were normalized by total antisense transcript as described previously [38]. Error bars represent standard deviation (SD). (D) Effect of rrp6l1-3 and rrp6l2-3 mutations on the level of H3K4me3 examined by ChIP using antibodies against H3K4me3 in the various regions of FLC. The level of H3K4me3 in rrp6l1/2 mutants was normalized to the level of H3K4me3 in wild type Col-0 plants. (E) AtRRP6L1 protein physically interacts with the FLC locus. ChIP assays were done using the rrp6l1-3 mutant complemented by a functional AtRRP6L1-TAP transgene. AtRRP6L1-TAP recruitment was normalized to wild type Ws, the background of the rrp6l1-3 mutant. The error bars in ChIP experiments represent the standard error of the mean and correspond to the difference between 2 biological replicates.
Figure 3.
Characterization of the ASL transcript.
(A) The diagram of the FLC gene and ASL RNA produced from the FLC locus in wild type plants. Gray boxes depict antisense transcript exons and gray lines indicate spliced regions. Alternatively spliced antisense transcript isoforms found by tiling RT-PCR. Arrowheads indicate position of primers used for RT-PCR amplification of ASL. F, H and 3′-end indicate that the regions used for RT-PCR amplification after RNA-IP (in Fig. 5A and B). The blue line depicts the COLDAIR transcript. (B) The ASL transcript is capped at the 5′end. RNA samples of Col-0 plants were treated with Terminator 5′-Phosphate-Dependent Exonuclease (TPE), which degrades uncapped RNA. (−) corresponds to the RNA sample before TPE treatment and (+) corresponds to the TPE treated sample. ACTIN 7 and rRNA were used as capped and un-capped controls for TPE treatment, respectively. (C) Expression of ASL in Col-0 plants at 10, 14, 18 and 21 days after germination of vegetative phase. Arrowheads indicate the two alternatively spliced isoforms of ASL. cDNA was synthesized using either antisense RNA specific primers or oligo-dT primers. TUBULIN 2 was used as an internal control and was amplified from the sample reverse transcribed with either tubulin 2-specific primers (second row) or the oligo-dT primers (bottom row). (D) α-amanitin treatment of Col-0 (11 days seedlings). No-T corresponds to non-treated plants, and 17-T corresponds to plants treated with α-amanitin for 17 hours. 17-T and 17-T′ indicate independent biological replicates.
Figure 4.
The level of ASL transcripts is decreased in rrp6l mutants.
(A) Expression of ASL RNA in rrp6l1-3, rrp6l2-3 and rrp6l1/2 double mutants. (B) The level of ASL transcript expression is restored in the rrp6l1-3 mutant complemented by functional AtRRP6L1-TAP transgene. ACTIN 7 was used as a loading control. Transgenic plants are in Col-0 ecotype. (C) Decay rate of ASL transcript in rrp6l1-1 mutants. α-amanitin treatment was performed for 0, 6 and 9 hours. The expression of antisense transcript was normalized to ACTIN 7.
Figure 5.
ASL transcript associates with AtRRP6L1 protein and H3K27 trimethylated regions and rrp6l1/2 mutants have decreased H3K27me3 and nucleosome density.
(A) ASL transcript directly associates with AtRRP6L1 protein and H3K27me3 regions of FLC. RNA-IP was performed using anti-AtRRP6L1 and anti-H3K27me3 antibodies to precipitate ASL RNA from wild type Col-0 plants. The regions used in RT-PCR (region H and the 3′end of ASL) are shown on Fig. 3A. No AB indicates no antibodies and is the negative control for RNA-IP. NT indicates no template and is the negative control for RT-PCR. (B) ASL transcript directly associates with AtRRP6L1 in rrp6l1-3 mutants complemented by functional AtRRP6L1-TAP transgene. Transgenic plants are in the Ws ecotype and Ws was used as negative control for RNA-IP. RNA-IP was performed using IgG antibodies to co-precipitate ASL RNA with RP6L1-TAP from wild type plants complemented with a transgene expressing RRP6L1-TAP. NT indicates no template and is the negative control for RT-PCR. (C) The level of H3K27me3 is decreased in rrp6l1/2 mutants. ChIP assays were performed using H3K27me3 antibodies. The level of H3K27me3 in rrp6l1/2 mutants was plotted relative to the level of H3K27me3 in Col-0 plants. The error bars in ChIP experiments represent the standard error of the mean and correspond to the difference between 2 biological replicates. (D) Nucleosome density is decreased in rrp6l1/2 mutants. Mnase-ChIP assays were performed using histone H3 antibodies. Nucleosomal density in rrp6l1/2 mutants was plotted relative to the level of nucleosomal density in Col-0 plants. The error bars in ChIP experiments represent the standard error of the mean and correspond to the difference between 2 biological replicates.