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The RNA exosome promotes transcription termination of backtracked RNA polymerase II

Nature Structural & Molecular Biology volume 21, pages 919–926 (2014)Cite this article

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Abstract

The exosome is an RNA-decay complex that constantly monitors transcription and contributes to post-transcriptional turnover of faulty mRNAs. Yet how nuclear RNA surveillance by the exosome is coordinated with transcription is still unknown. Here we show that the RNA exosome of Schizosaccharomyces pombe can target the transcription machinery by terminating transcription events associated with paused and backtracked RNA polymerase II (RNAPII); this is contrary to the notion that the exosome acts exclusively on RNAs that have been released by RNAPII. Our data support a mechanism by which RNAPII backtracking provides a free RNA 3′ end for the core exosome, which results in transcription termination with concomitant degradation of the associated transcript. These findings uncover a mechanism of cotranscriptional RNA surveillance whereby termination of transcription by the exosome prevents formation of aberrant readthrough RNAs and transcriptional interference at neighboring genes.

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Figure 1: Cells deficient in core exosome subunits, but not the TRAMP complex, accumulate readthrough RNAs.
Figure 2: Widespread production of 3′-extended transcripts in exosome-depleted cells.
Figure 3: The core exosome promotes termination of RNAPII transcription.
Figure 4: Transcription-dependent recruitment of core exosome subunits to genes.
Figure 5: The 3′-5′ exonucleolytic activity of Dis3 and the exosome central channel are required for transcription termination.
Figure 6: Transcription termination by the exosome is mechanistically linked to RNAPII backtracking.
Figure 7: Model of cotranscriptional RNA surveillance by the RNA exosome by competition with 3′-end processing.

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Acknowledgements

We thank M. Bühler and P.-E. Jacques for helpful discussions; M. Yamamoto (University of Tokyo) and D. Hermand (University of Namur) for strains; M. Durant and E. Lapointe (both at Laboratoire de Génomique Fonctionnelle de l'Université de Sherbrooke) for ChIP-seq libraries; M. Luetzelberger (University of Braunschweig) for the tetracycline-inducible expression vector; the sequencing platform of the McGill University and Génome Québec Innovation Centre; and S. Abou Elela, E. Lei and A. Morillon for critical reading of the manuscript. This work was supported by funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) to F.B., from a Wellcome Trust Senior Investigator Award to J.B. and from the UK Medical Research Council to S.M. J.-F.L. was supported by an Alexander Graham-Bell Doctoral Scholarship from NSERC. F.B. is supported as a Canada Research Chair in Quality Control of Gene Expression.

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Author notes

  1. Jean-François Lemay and Marc Larochelle: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, RNA Group, Université de Sherbrooke, Sherbrooke, Québec, Canada

    Jean-François Lemay, Marc Larochelle & François Bachand

  2. Medical Research Council Clinical Sciences Centre, Imperial College London, London, UK

    Samuel Marguerat

  3. Department of Genetics, Evolution and Environment, University College London, London, UK

    Sophie Atkinson & Jürg Bähler

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  1. Jean-François Lemay
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Contributions

J.-F.L., M.L. and F.B. conceived and designed the research project; J.-F.L. and M.L. performed the experiments; S.M. and S.A. prepared the RNA-seq libraries in collaboration with J.B.; S.M. performed computational analyses of RNA-seq and ChIP-seq experiments; J.-F.L., M.L., S.M. and F.B. analyzed the data; F.B. wrote the manuscript; and all authors discussed the results and commented on the manuscript.

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Correspondence to François Bachand.

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Integrated supplementary information

Supplementary Figure 1 Exosome depletion results in the production of readthrough RNAs.

(a) Western blot analysis of extracts prepared from wild-type (lanes 1-2) and Pnmt1-dis3 (lanes 3-4) cells that were grown in the absence (-) or presence (+) of thiamine. (b) Northern blot analysis of RNA prepared from wild-type (lanes 1-2), Pnmt1-rrp41 (lanes 3-4), and Pnmt1-dis3 (lanes 5-6) cells that were grown in the absence (-) or presence (+) of thiamine. Note the thiamine-dependent increase in 5.8S rRNA precursor (7S rRNA) and concomitant decrease in mature 5.8S rRNA in Dis3- and Rrp41-depleted cells (lanes 4 and 6, respectively), but not in thiamine-treated wild-type cells (lane 2). (c) Schematic of the RNase H cleavage assay used to detect 3′-extension of the snR3 snoRNA. (d) Equal amounts of total RNA prepared from the indicated strains grown in the absence (-) or in the presence (+) of thiamine were treated with RNase H in the presence of DNA oligonucleotides complementary to sequences located beyond the polyadenylation site (poly(A)) of snR3. The positions of the DNA oligonucleotides used are shown in the schematic representation (c). Read-through snR3 transcripts (3′-ext) were detected using a strand-specific riboprobe complementary to the RNase H-cleaved product. Size markers (nt) are indicated on the right. The 5S rRNA was used as a loading control. (e) Binding of Pcf11-TAP relative to RNAPII occupancy was calculated for three independent regions of the pma1 gene (shown on the schematic above the graph) in wild-type (WT) and Pnmt1-dis3 cells grown in the presence of thiamine. Percent inputs for Pcf11-TAP were corrected for Pol II occupancy along pma1 using the CTD-specific antibody (8WG16). Bars indicate the range of measured values from two independent experiments.

Supplementary Figure 2 Readthrough RNAs detected in exosome-depleted cells are cleaved and polyadenylated downstream of motifs associated with 3′-end processing and can result in the production of chimeric transcripts.

(a) Schematic summarizing oligo d(T)-primed 3′ RACE analyses of polyadenylation site positions for hsp9 in wild-type and Dis3-depleted cells. Red arrows indicate the position of polyadenylation sites for the regular length hsp9 mRNA in both wild-type and Dis3-depleted cells, whereas blue arrows indicate the polyadenylation sites for the read-through hsp9 mRNAs detected in Dis3-depleted cells. (b) Shown are the nucleotide positions of poly(A) sites identified by 3′ RACE for regular length (red) and read-through (blue) mRNAs in the hsp9 gene. The underlined DNA sequences represent motifs known to be over-represented in regions -10 to -30 relative to cleavage site1,2, including the AATAAA hexamer. (c) Northern analysis of total RNA prepared from wild-type (lanes 1 and 3) and Pnmt1-dis3 (lanes 2 and 4) cells that were supplemented with thiamine. The blot was hybridized with an antisense RNA probe complementary to SPCC1442.13c (lanes 1-2; Probe A) and SPCC1442.14c (lanes 3-4; Probe B). The position of the SPCC1442.14c poly(A) site was identified by RNA-seq2. Note that the SPCC1442.14c-SPCC1442.13c chimeric transcripts are recognized using both RNA probes. The 25S rRNA was used as a loading control.

Supplementary Figure 3 The Rrp6 exoribonuclease does not extensively contribute to the suppression of readthrough transcripts.

(a) Northern blot analysis of total RNA prepared from wild-type (lanes 1 and 4), Pnmt1-dis3 (lane 2), and rrp6Δ (lane 3) cells that were grown in minimal media supplemented with thiamine (lanes 1-2) or rich media (YES; lanes 3-4). The blots were analyzed using strand-specific antisense RNA probes. The 25S rRNA was used as a loading control. (b) Northern analysis of RNase H-cleaved snR3 3’-extensions as described in Supplementary Fig. 1c using total RNA prepared from cells described in (a).

Supplementary Figure 4 Readthrough transcription by RNAPII in exosome-deficient cells.

(a) Average distribution of RNAPII density in the indicated strains, as determined by analysis of ChIP-seq results for all (n=7005), mRNA (n=5123), snoRNA (n=53), and snRNA (n=7) genes. Orange rectangles represent open reading frames (mRNA genes) and coding sequences (snoRNA and snRNA genes) and black arrows 500-bp of upstream and downstream flanking DNA sequences. (b) Average distribution of RNAPII density in the indicated strains, as determined by analysis of ChIP-seq results for (i) tandem genes that are more than 200-bp apart (n=1425), (ii) tandem genes that are more than 400-bp apart (n=855), (iii) convergent genes that are more than 200-bp apart (n=452), (iv) convergent genes that are more than 400-bp apart (n=208). Orange rectangles represent open reading frames and black arrows 500-bp of upstream and downstream flanking DNA sequences. (c) Schematic of the ura4 transgene that was integrated into wild-type and Pnmt1-dis3 strains. The termination element of the snR99 gene3 was inserted upstream of the ura4 gene. Arrows indicate the position of the primer pairs used for RNA and ChIP analyses in (g) and (h), respectively. (d) Northern blot of total RNA prepared from wild-type cells that have a normal ura4 gene (lanes 1 and 3) or a ura4 gene in which the snR99 terminator (snR99T) was inserted upstream (lanes 2 and 4), as described in (c). Note that ura4 mRNAs are not detected in cells that express ura4 with the snR99 terminator (lane 2). Read-through snR99T-ura4 transcripts were detected at low levels on a long exposure in wild-type cells (lane 4). (e) Ten-fold serial dilutions of wild-type cells transformed with the empty vector, the normal ura4 construct, or the snR99T-ura4 construct were spotted on uracil-supplemented (left) or uracil-free (right) media. Cells with the snR99T-ura4 construct do not grow on uracil-free medium. (f) Northern analysis of RNA from the indicated strains that were previously transformed with the snR99T-ura4 construct and that were grown in the absence (-) or presence (+) of thiamine. The arrow points to the snR99T-ura4 chimeric RNA, which accumulates in Dis3-depleted cells (lane 4). (g) RT-qPCR analysis of snR99T-ura4 RNA levels using the strains described in (f). (h) ChIP analysis of RNAPII at the promoter and ORF regions of the snR99T-ura4 construct. RNAPII density values are relative to cells grown in the absence of thiamine. Note the greater RNAPII density in the ORF region of ura4 relative to the promoter region, indicative of increased transcription through the snR99 terminator in Dis3-depleted cells. Error bars, s.d. (n = 3 biological replicates from independent cell cultures). P values are from two-tailed Student's t test.

Supplementary Figure 5 Expression and growth phenotypes of D166N (endo–), D516N (exo–) and D166N D516N (endo– exo–) versions of S. pombe Dis3.

(a) Amino acid sequence alignment of S. pombe and S. cerevisiae Dis3 using Clustal Omega. Identical and similar residues are highlighted in black and gray, respectively. The PIN and RNB domains of S. pombe and S. cerevisiae Dis3 are boxed in red and blue, respectively. To create an endonucleolytic mutant of S. pombe Dis3, aspartic acid (D) residue 166 within the PIN domain was substituted to asparagine (D166N), a substitution homologous to D171N of S. cerevisiae Dis3 that results in a version of Dis3 devoid of endonucleolytic activity4. To create an exonucleolytic mutant of S. pombe Dis3, aspartic acid (D) residue 516 within the RNB domain was substituted to asparagine (D516N), a substitution homologous to D551N of S. cerevisiae Dis3 that results in a version of Dis3 devoid of exonucleolytic activity4. (b) Western blot analysis of extracts prepared from wild-type cells (lanes 1-2) as well as from Pnmt1-dis3 cells in which an empty vector (lanes 5-6) or constructs that express wild-type Dis3 (lanes 7-8), endo mutant (lanes 9-10), and exo mutant (lanes 11-12) versions of Dis3. Cells grown in the presence (+) of thiamine were depleted for endogenous Dis3 (lanes 4, 6, 8, 10, and 12). (c) Ten-fold serial dilutions of wild-type and Pnmt1-dis3 cells as well as Pnmt1-dis3 cells that were transformed with an empty vector (EV) or a construct that expresses wild-type Dis3 (WT) were spotted on thiamine-free (left) or thiamine-containing (right) minimal media. (d) Ten-fold serial dilutions of wild-type cells as well as Pnmt1-dis3 cells that were transformed with an empty vector (EV) or constructs that express endonuclease-deficient (endo-) Dis3, exonuclease-deficient (exo-) Dis3, and the endo- and exo-deficient double mutant (endo- exo-). Cells were spotted on thiamine-free (left) or thiamine-containing (right) minimal media. The growth phenotypes of the S. pombe Dis3 endo-, exo-, and double endo- exo- mutant strains showed the same phenotypes of the corresponding S. cerevisiae mutants strains4,5: the endo mutant did not show growth defects, whereas growth of the exo mutant was impaired. Introducing the D166N substitution of the endonuclease domain into the exo mutant (endo- exo- double mutant) exacerbated the growth phenotype of the single exo mutant, consistent with the view that both catalytic activities are required for cell viability4,5. (e) Northern analysis of the pma1 mRNA using the indicated strains grown in the presence of thiamine. The arrow indicates the read-through pma1 transcript.

Supplementary Figure 6 The central channel is essential for viability and snoRNA processing but does not impair exosome assembly in S. pombe.

(a) Amino acid sequence alignment of S. pombe (Sp) and S. cerevisiae (Sc) Rrp41 using Clustal Omega. Identical and similar residues are highlighted in black and gray, respectively. The central channel of the S. pombe exosome was occluded by inserting an 11-amino acid sequence (GESEGESEGLE) between serine-62 and lysine-63. This insertion is similar in length and amino acid composition to a previously described insertion that was shown to physically occlude the central channel of the S. cerevisiae RNA exosome6. (b) Ten-fold serial dilutions of wild-type (WT) and Pnmt1-rrp41 cells that were transformed with an empty vector (EV) or constructs that express wild-type (WT) or channel-occluded (CO) Rrp41. Cells were spotted on thiamine-free or thiamine-containing minimal media. The channel-occluded version of Rrp41 caused growth defect similar to Pnmt1-rrp41 cells transformed with the empty vector. As expected, a wild-type version of Rrp41 restored the growth defect resulting from depletion of endogenous Rrp41. (c) Total RNA prepared from wild-type (lanes 1-2) and Pnmt1-rrp41 (lanes 3-8) strains was treated with RNase H in the presence of a DNA oligonucleotide complementary to H/ACA class snoRNA snR99. RNase H reactions were performed in the presence (+) or absence (−) of oligo(dT). 5S rRNA was used as a loading control. Expression of a channel-occluded (CO) version of Rrp41 accumulated levels of 3′-extended polyadenylated snR99 precursors (lanes 5-6) similar to Pnmt1-rrp41 cells transformed with the empty vector (EV) control (lanes 3-4). (d) A channel-occluded version of Rrp41 does not interfere with core exosome assembly. Immunoblot analysis of whole-cell extracts (WCE; lanes 1–3) and IgG-sepharose precipitates (IP; lanes 4–6) prepared from wild-type (WT) cells transformed with an empty vector (EV) or from Pnmt1-rrp41 cells expressing a TAP-tag version of Csl4 transformed with constructs expressing either wild-type (WT; lanes 3 and 6) or channel-occluded (CO; lanes 2 and 5) versions of Rrp41. Cells were grown in the presence of thiamine to repress endogenous Rrp41. Immunoprecipitation of the exosome “cap” protein, Csl4-TAP, in the presence of wild-type (lane 6) or channel-occluded (lane 5) versions of Rrp41 recovered similar levels of Dis3, which is located at the bottom of the core exosome, supporting proper exosome assembly.

Supplementary Figure 7 Transcription termination by the RNA exosome is mechanistically linked to RNAPII backtracking and pausing.

(a-c) A Tfs1 mutant that lacks transcript cleavage stimulatory activity and inhibits unstimulated cleavage results in a more pronounced suppression of read-through RNAs relative to the tfs1 deletion in Dis3-depleted cells. (a) The S. pombe transcription elongation factor Tfs1 possesses a highly conserved acidic loop important for cell viability and transcript cleavage. Sequence alignments of the transcription elongation factors Tfs1 of S. pombe and Dst1p (TFIIS) of S. cerevisiae. Arrows indicate the acidic amino acids involved in stimulating transcript cleavage inside the catalytic center of RNAPII. In order to abolish transcript cleavage, the conserved Asp274-Glu275 dipeptide of the acidic loop were changed to alanine residues, as described previously in S. cerevisiae7. (b) Analysis of cell growth by optical density measurements (OD600). Pnmt1-dis3 tfs1Δ cells that were previously transformed with DNA constructs expressing wild-type or mutant (D274A E275A) versions of Tfs1 under the control of a tetracycline-inducible promoter, as well as the empty vector (EV) control, were grown in media containing 7.5 μM of the non-metabolic inducer anhydrotetracycline hydrochloride (+AhTET; right) or in mock-treated media (-AhTET; left). Samples were prepared in triplicate and cell growth was monitored every 10 min for 48 h. Inducing the expression of Tfs1 D274A E275A (tfs1 mutant) causes severe growth defect, as shown previously in S. cerevisiae. (c) real-time RT-qPCR analysis of hsp9 read-through and total mRNA using RNA obtained from cell grown as described in Fig. 6f. The hsp9 3′-extensions/total RNA ratio was arbitrarily set to 1.0 for the Pnmt1-dis3 + EV strain. Error bars, s.d. (n = 3 biological replicates from independent cell cultures). P values are from two-tailed Student′s t test. (d-f) Termination of transcription by the exosome requires RNA polymerase II pausing. (d) Schematic of wild-type and mutant ura4 constructs used in (e) and (f). Site-determining elements (SDE) 1-2 and downstream sequence elements (DSE) important for RNAPII pausing 8 are shown. The red arrows indicate the position of the PCR primers used to analyze RNAPII density at the 3′ end of ura4. (e) ChIP analysis of RNAPII at the 3′ end of the wild-type and mutant ura4 gene in wild-type cells. Disruption of transcriptional pause signals (mutant) resulted in transcription termination defects, as measured by the 4-fold increase in RNAPII density at the 3′ end of the mutant ura4 gene relative to wild-type ura4, consistent with previous findings8. (f) Wild-type and mutant ura4 constructs were inserted into the Pnmt1-dis3 conditional strain, and RNAPII density was measured at the 3′ end of ura4 by ChIP assays using cells cultured in the presence and absence of thiamine. A thiamine-dependent increase in RNAPII density was detected at the 3′ end of the wild-type ura4 gene (WT ura4 3′) in Pnmt1-dis3 cells (red) relative to wild-type cells (blue), consistent with termination defects in Dis3-depleted cells. In contrast, read-through polymerases that result from loss of exosome function (red) were no longer detected at the 3′ end of the pause-defective ura4 mutant (mutant ura4 3′). RNAPII density values are relative to cells grown in the absence of thiamine. (e-f) Error bars, s.d. (n = 3 biological replicates from independent cell cultures). P value is from two-tailed Student′s t test.

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Supplementary Figures 1–7 and Supplementary Table 1 (PDF 5178 kb)

Supplementary Data Set 1

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Lemay, JF., Larochelle, M., Marguerat, S. et al. The RNA exosome promotes transcription termination of backtracked RNA polymerase II. Nat Struct Mol Biol 21, 919–926 (2014). https://doi.org/10.1038/nsmb.2893

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