Figure 1.
Purification of exosome complexes.
Silver-stained SDS-PAGE of proteins co-immunoprecipitated with RRP41-myc. Similar results were obtained when GFP-tagged RRP41 was used as bait. The three main bands observed in mock IP and also present in RRP41-myc IP correspond to immunoglobulins.
Table 1.
Proteins co-purified with tagged RRP41 and identified by LC-MS/MS.
Figure 2.
HEN2 proteins are restricted to the green lineage.
Phylogenetic tree of the MTR4/SKI family of RNA helicases. The basic branch that separates HEN2 homologues from MTR4 and SKI2 homologues is detected with 1000/1000 bootstraps. Protein sequences were retrieved from metazome, phytozome and JGI databases and aligned with ClustalX. Dark red, vertebrates; light red, other eumetazoa; pink, M. brevicollis (Choanozoa); blue, B. natans (Rhizaria); orange, N. gruberi (Heterobolosea); yellow, S. cerevisiae and S. pombe (Fungi); olive, F. cylindrus, P. tricornutum, T. pseudonana; P. cinammomi, P. sojae (Heterokonta); light green, green algae; green, mosses; blue-green, grasses; dark green, dicotyledons. Scale bar = 0.05 amino acid substitutions per site.
Figure 3.
MTR4 and HEN2 have distinct localization patterns.
Distribution of GFP-fusion proteins in root cells of stable Arabidopsis transformants. No, Nucleolus; Np, Nucleoplasm; Cp, Cytoplasm. Scale bars 5 µm.
Table 2.
Proteins co-purified with MTR4-GFP and identified by LC-MS/MS.
Table 3.
Proteins co-purified with HEN2-GFP at 50 mM and 150 mM ionic strength, and identified by LC-MS/MS.
Figure 4.
Loss of HEN2 results in over-accumulation of selected exosome targets.
Steady-state levels of exosome targets selected from [5], (see also Fig. S8) in hen2 and mtr4 mutant seedlings were determined by qRT-PCR. Samples from an inducible RRP41 RNAi line grown in absence (RRP41 ctrl) or presence (RRP41 RNAi) of estradiol were included as controls. The histogram shows the fold change relative to wild type. mtr4-1 in red, mtr4-2 in orange, hen2-2 in light green, hen2-4 in dark green, RRP41 control in light grey, RRP41 RNAi in dark grey. Error bars = SD in three biological replicates.
Figure 5.
hen2 mutants accumulate short transcripts derived from mRNA genes.
qRT-PCR analysis. A diagram of the genomic locus indicated by the respective AGI number is shown at the top of each panel. Annotated mRNA genes are represented as arrows with dark blue boxes for the CDS, light blue boxes for 3′ and 5′ UTRs, and a light blue line for introns. Red bars above the diagram represent probes detected in the tiling analysis. Green arrows above or below the diagram depict the location of qRT-PCR primers. The corresponding qRT-PCR results for each primer pair are given as fold-change relative to WT in the histograms below each diagram. mtr4-1 in red, mtr4-2 in orange, hen2-2 in light green, hen2-4 in dark green, RRP41 control in light grey, RRP41 RNAi in dark grey. Error bars = SD in three biological replicates.
Figure 6.
Accumulation of 3′ extended mRNAs in hen2 mutants.
qRT-PCR experiments to test the upregulation of regions that mapped within the body and beyond annotated 3′ ends of mRNAs. Please see legend of Fig. 5 for a detailed explanation of the diagrams.
Figure 7.
hen2 mutants accumulate incompletely spliced transcripts.
A qRT-PCR. The diagram shows the At1g79170 locus. The At1g79170 mRNA is represented as an arrow with dark blue boxes for the CDS, light blue boxes for 3′ and 5‚ UTRs, and a light blue line for introns. Red bars above the diagram represent probes detected in the microarray analysis. Green arrows above or below the diagram depict the location of qRT-PCR primers. The corresponding qRT-PCR results for each primer pair are given as fold-change relative to WT in the histograms below each diagram. Please note the scales. mtr4-1 in red, mtr4-2 in orange, hen2-2 in light green, hen2-4 in dark green, RRP41 control in light grey, RRP41 RNAi in dark grey. Error bars = SD in three biological replicates. B 3′ RACE PCR. The diagram shows the At1g79170 locus. Forward primers used for 3′ RACE-PCR on oligo-dT-primed cDNA are shown as green arrows above the diagram. A negative stain of PCR products separated on a 1.5% agarose gel is shown on the bottom. The upper band marked by a star corresponded to the fully spliced mRNA as depicted by the long orange arrow below the diagram. The lower band marked by two stars corresponded to transcripts depicted by the short orange arrow, all of which comprised the unspliced donor site of the first exon/intron junction. The 3′ extremities of these transcripts were located at or upstream of the 3′ acceptor site and were polyadenylated (see Fig. S10).
Figure 8.
hen2 mutants accumulate regions of excised introns.
qRT-PCR experiments to validate the upregulation of regions located within introns. Primers pairs V62a, V64a/b and V70ab are exon spanning and produce amplicons only from spliced mRNAs under the conditions used for qRT-PCR. Please refer to the legend of Fig. 5 for a detailed explanation of the diagrams.
Figure 9.
hen2 mutants accumulate snoRNA precursors.
qRT-PCR. A diagram of the genomic locus with the indicated snoRNA genes is shown at the top of each panel. Individual snoRNA genes are represented as yellow arrows. Red bars above the diagram represent probes detected in the tiling analysis. Green arrows above or below the diagram depict the location of qRT-PCR primers. The corresponding qRT-PCR results for each primer pair are given as fold-change relative to WT in the histograms below each diagram. mtr4-1 in red, mtr4-2 in orange, hen2-2 in light green, hen2-4 in dark green, RRP41 control in light grey, RRP41 RNAi in dark grey. Error bars = SD in three biological replicates.
Figure 10.
Loss of HEN2 results in accumulation of polyadenylated antisense transcripts.
3′ RACE-PCR. A diagram of the At5g44306 locus is shown at the top. A blue arrow represents the At5g44306 mRNA. Red bars above the diagram represent probes detected in the tiling analysis. The location of the primer used for 3′ RACE PCR is indicated by a green arrow above the diagram. Each of the orange horizontal bars below the diagram represents a polyadenylated clone obtained from the indicated sample. 28 of 32 clones obtained from hen2 samples and 3 of 32 clones obtained from mtr4 samples corresponded to antisense transcripts of 67 to 208 nt lenght. 0 of 32 clones obtained from WT samples corresponded to the target sequence.
Figure 11.
Degradation of non-ribosomal exosome targets depends largely on HEN2.
The transcriptomes of WT, hen2-4 and mtr4-1 plants were compared by whole genome microarrays. The histogram shows the total number of regions that were, as compared to wild type, overaccumulated in hen2 or mtr4 samples, respectively. A complete list of the upregulated regions can be found in Tables S4, S5, S6, S7, S8, S9, S10, S11.
Figure 12.
Effect of mtr4 and hen2 mutations on posttranscriptional silencing.
Diagrams show the proportion of plants that undergo systemic silencing of a GUS (panels A and B) or AGO1 (panel C) reporter transgene in the indicated mutants and backgrounds. The color code indicates the intracellular localization of the mutated proteins: red marks the nucleolar proteins; green marks nucleoplasmic proteins, and blue mark cytoplasmic proteins. A. Loss of MTR4 has only a marginal effect on GUS S-PTGS. The effect of the mtr4-2 mutation on silencing of a GUS reporter transgene was tested in the Hc1/Col reporter system as described in [51]. For comparison, we included previously published data from [49] that show the effects of compromised 5′-3′ exoribonucleases XRN2 (nucleolar), XRN3 (nucleoplasmic) or XRN4 (cytoplasmic) in the same reporter system. B. Loss of HEN2 triggers silencing of a GUS PTGS reporter. The effect of hen2-1 (in Ler) on GUS-PTGS was tested in an Hc1-reporter line backcrossed to Ler. Please note that the Hc1/Ler reporter line does not trigger silencing of the GUS reporter in WT. xrn4 in Hc1/Ler was used as a positive control. C. Loss of HEN2 triggers co-suppression of AGO1. To further confirm the role of HEN2 as silencing suppressor, WT, hen2-2 and xrn4 plants (in Col) were transformed with a pAGO1::AGO1 construct that triggers cosuppression of the endogenous AGO1 gene.