System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organization and function of RNA–protein complexes

Large RNA-binding complexes play a central role in gene expression and orchestrate production, function, and turnover of mRNAs. The accuracy and dynamics of RNA–protein interactions within these molecular machines are essential for their function and are mediated by RNA-binding proteins (RBPs). Here, we show that fission yeast whole-cell poly(A)+ RNA–protein crosslinking data provide information on the organization of RNA–protein complexes. To evaluate the relative enrichment of cellular RBPs on poly(A)+ RNA, we combine poly(A)+ RNA interactome capture with a whole-cell extract normalization procedure. This approach yields estimates of in vivo RNA-binding activities that identify subunits within multiprotein complexes that directly contact RNA. As validation, we trace RNA interactions of different functional modules of the 3′ end processing machinery and reveal additional contacts. Extending our analysis to different mutants of the RNA exosome complex, we explore how substrate channeling through the complex is affected by mutation. Our data highlight the central role of the RNA helicase Mtl1 in regulation of the complex and provide insights into how different components contribute to engagement of the complex with substrate RNA. In addition, we characterize RNA-binding activities of novel RBPs that have been recurrently detected in the RNA interactomes of multiple species. We find that many of these, including cyclophilins and thioredoxins, are substoichiometric RNA interactors in vivo. Because RBPomes show very good overall agreement between species, we propose that the RNA-binding characteristics we observe in fission yeast are likely to apply to related proteins in higher eukaryotes as well

The Sm Complex Is Required for the Processing of Non-Coding RNAs by the Exosome

<div><p>A key question in the field of RNA regulation is how some exosome substrates, such as spliceosomal snRNAs and telomerase RNA, evade degradation and are processed into stable, functional RNA molecules. Typical feature of these non-coding RNAs is presence of the Sm complex at the 3′end of the mature RNA molecule. Here, we report that in <i>Saccharomyces cerevisiae</i> presence of intact Sm binding site is required for the exosome-mediated processing of telomerase RNA from a polyadenylated precursor into its mature form and is essential for its function in elongating telomeres. Additionally, we demonstrate that the same pathway is involved in the maturation of snRNAs. Furthermore, the insertion of an Sm binding site into an unstable RNA that is normally completely destroyed by the exosome, leads to its partial stabilization. We also show that telomerase RNA accumulates in <i>Schizosaccharomyces pombe</i> exosome mutants, suggesting a conserved role for the exosome in processing and degradation of telomerase RNA. In summary, our data provide important mechanistic insight into the regulation of exosome dependent RNA processing as well as telomerase RNA biogenesis.</p></div

The exosome mutants with the non-functional Sm site accumulate 3′extended poly(A)+ telomerase RNA.

<p>A) Schematic diagram showing the sequence and position of the Sm site and its mutated variants in the plasmid encoded <i>TLC1</i> RNA. B) Analysis of exosome mediated <i>TLC1</i> processing in the Sm site mutants. In this experiment parent strains with either <i>RRP47</i> (lanes 1, 3, 5 and 7) or <i>rrp47</i>Δ (lanes 2, 4, 6 and 8) alleles were used, in which the endogenous <i>TLC1</i> gene has been deleted and replaced with both <i>TLC1</i> expressed from a <i>URA3</i> marked plasmid and a <i>LEU2</i> marked plasmid encoding either <i>TLC1</i> (lanes 1–2) or mutated Sm site variants (lanes 3–8) as described in (B). RNA was isolated from after shuffling out the WT pRS316-<i>TLC1</i> (<i>URA3</i>) plasmid on 5-FOA media, resolved on a 5% acrylamide gel, and visualized using the probes for <i>TLC1</i>, U1 and <i>ADH1</i>. Positions of poly(A)+ and poly(A)− <i>TLC1</i> RNAs are indicated with grey boxes. 18S rRNA is indicated. C) Schematic diagram describing positions of the two major RNase H products and the probe used for detecting by Northern blotting. D) Analysis of the <i>TLC1</i> 3′ends by RNase H experiment. RNase H treated total RNA using either oligo #688 (lanes 2 and 4) or oligos #2008 together with oligo dT (lanes 1, 3 and 5) from WT, <i>rrp47</i>Δ and <i>tlc1</i> 4C5C strains described in (B) were analysed by Northern blotting. Two major RNase H products corresponding to the precursor and mature RNAs are indicated. The lower panel shows Methylene-Blue stained RNA as a loading control.</p

Telomerase RNA can be trimmed by the exosome complex <i>in vitro</i>.

<p>A) Schematic diagram explaining the experimental set up of the <i>in vitro</i> telomerase RNA processing reaction. Exosome complex purified as described in (A) was incubated with the Est2 associated telomerase RNA isolated on IgG agarose from cells expressing ProteinA-Est2 (YLV124, 125, and 126 strains). B) The exosome processes but does not degrade the longer form of telomerase RNA in the presence of a functional Sm site <i>in vitro</i>. Telomerase RNA purified from <i>rrp47</i>Δ (YLV124), <i>tlc1sm4C5C</i>, <i>rrp47</i>Δ (YLV125) and <i>TLC1</i> (YLV126) was incubated with the exosome complex for 90 min and reaction products were analysed by RT-PCR using oligos F (#2492) and R1 (#2489) (lanes 2–7), F and R2 (#2493) (lanes 8–14). PCR conditions were optimized and reactions were performed for 25 cycles, PCR products were resolved on a 2% agarose gel. C, D) Relative RNA levels were measured by quantification of PCR band intensities from lanes 2–7 and 8–13 in (B) using ImageJ v1.32 software. Error bars are from three independent repetitions.</p

An Sm site is required for the processing of the U1 RNA precursor by the exosome complex.

<p>A) Schematic diagram describing the organization of plasmid-borne <i>SNR19</i> and <i>SNR19</i>Δ<i>192–507</i> genes, which drive the expression of U1 and U1Δ192–507 RNA respectively. Endogenous <i>SNR19</i> was deleted and replaced by plasmids encoding <i>SNR19</i> and either <i>SNR19</i>Δ<i>192–507</i> or <i>SNR19</i>Δ<i>192–507sm,</i> that lacks an Sm site. The plasmid-borne copy of full length <i>SNR19</i> is under the control of a galactose inducible promoter (<i>GAL10p</i>), such that expression of the WT gene can be shut down by culturing cells in glucose media, while <i>SNR19</i>Δ<i>192–507</i> and <i>SNR19</i>Δ<i>192–507sm</i> are expressed constitutively from the endogenous <i>SNR19</i> promoter (<i>SNR19p</i>). B) Processing of U1 RNA upon disruption of the exosome/Sm-mediated processing pathway. Northern blot analysis of U1 processing in U1(Δ192–507)sm, GAL::U1 (YF2081); U1(Δ192–507)sm, GAL::U1, <i>rrp47</i>Δ (YLV48); U1(Δ192–507) (YF2088) and U1(Δ192–507), <i>rrp47</i>Δ (YLV68) (lanes 1–4 respectively). Positions of U1(Δ192–507) precursor and U1(Δ192–507) mature RNAs are indicated. Bands corresponding to the precursor RNA are marked with asterisks. 18S RNA is also shown. C) U1 precursor RNA does not compensate for the function of mature U1 RNA in pre-mRNA splicing. Splicing of <i>RP51A</i> RNA was analyzed by northern blot of total RNA from U1(Δ192–507)sm, GAL::U1 (YF2081); or U1(Δ192–507)sm, GAL::U1, <i>rrp47</i>Δ (YLV48). Cells were pre-grown on galactose media to OD = 0.5 to allow for expression of full length U1 from the <i>GAL10</i> promoter (shown in (A)). Logarithmic cultures were maintained for a further 10 hours in media containing galactose (lanes 1 and 3) or glucose (lanes 2 and 4). Positions of spliced <i>RP51A</i> and its un-spliced precursor are indicated. Both forms of <i>RP51A</i> RNA (spliced and un-spliced) appear as double bands as previously reported. To control for loading, RNA was also probed for <i>ADH1</i>. D) Quantification of splicing efficiency of Sm mutated U1 RNA relative to WT. The splicing efficiency observed in the experiments described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065606#pone-0065606-g005" target="_blank">Figure 5C</a> was calculated as a ratio of spliced/un-spliced <i>RP51A</i> RNA precursor and normalized for the WT value.</p

A hypothetical model describing possible role for Sm binding in the exosome-mediated processing of non-coding RNAs.

<p>Non-coding RNA transcripts recognized by the nuclear exosome are degraded from their 3′ end (1). In the case of substrates that contain an Sm binding site the exosome trims along the RNA until it encounters the bound Sm complex. The Sm complex blocks the progression of the exosome (2) resulting in a processed, mature RNA. When the pathway is functional, binding of the Sm complex defines the processing mode of the exosome and protects Sm-containing RNA substrates from degradation. Removal of the Sm site or of Sm proteins results in a non-functional pathway: the blockade of the exosome progression is compromised, resulting in RNA degradation.</p

Poly(A)+ <i>TLC1</i> RNA is not functional in telomere elongation.

<p>A) Telomere length analysis by Southern blot. Genomic DNA was digested with <i>Xho</i>I and resolved on a 0.8% agarose gel. Southern blot analysis was performed using a 3′-end-digoxigenin-labeled oligonucleotide (TGTGGG)₄. Terminal fragments of Y′-element containing telomeres appear as a diffuse band as was previously described. Larger fragments detected by this probe correspond to non-telomeric poly(AC)_n repeats and non-Y′ containing telomeres. B) Analysis of the <i>TLC1</i> RNA from the strains described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065606#pone.0065606.s006" target="_blank">Figure S6</a> after 4^th re-streak. C) Est2 was purified via an N-terminal ProteinA tag from <i>rrp47</i>Δ (YLV124) (lane 1), <i>tlc1sm4C5C</i>, <i>rrp47</i>Δ (YLV125) (lane 2) and <i>TLC1</i> (YLV126) strains (lane 3), and the bead-bound fractions were analysed by immunoblotting using an anti-PAP antibody that can recognizes the ProteinA module. A mock purification was performed with YF336 strain (lane 4). D) RNA that co-immunoprecipitated on IgG beads with ProteinA-Est2 (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065606#pone-0065606-g003" target="_blank">Figure 3C</a>) from <i>tlc1sm4C5C</i>, <i>rrp47</i>Δ (YLV125) (lane 1), <i>TLC1</i> (YLV22) (lane 2) and non-tagged WT control (YF336) (lane 3) strains was analysed by RT PCR using oligonucleotides specific to the coding region of <i>TLC1</i> RNA (F(#2492) and R2(#2493)). E) Telomere extension assay. Telomerase complexes isolated from YLV125 (lane 1), YLV126 (lane 2) and YF336 (lane 3) strains described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065606#pone-0065606-g003" target="_blank">Figure 3C</a> were incubated with γ-labelled telomeric oligonucleotide (+1 oligo) in the presence of dTTP and [α-³²P]dGTP, reaction products (labelled with a bracket) were resolved on a 20% acrylamide, 7M urea gel.</p

Exosome mutants accumulate poly(A)+ telomerase RNA.

<p>A) Schematic diagram describing the positions of <i>TLC1 (S. cerevisiae)</i> and <i>TER1 (S. pombe)</i> probes for telomerase RNA. Probes are depicted as black bars. B) Accumulation of poly(A)+ <i>TLC1</i> RNA in <i>S. cerevisiae</i> exosome mutant strains. Northern blot analysis of total RNA from WT (YF336), lane 1; <i>rrp47</i>Δ (YF1465), lane 2; WT (YF1444), lane 3 and <i>rrp6</i>Δ (YSB2244), lane 4. RNA was resolved on a 5% PAGE and <i>TLC1</i> RNA was visualized with the probe depicted in (A). Positions of poly(A)+ and poly(A)− <i>TLC1</i> RNAs are indicated with grey boxes. Methylene-Blue stained 18 and 25S rRNAs are shown below and serve as a loading control. C) Exonucleolytic activity of the exosome is required for the processing of poly(A)+ <i>TLC1</i> RNA. Northern blot analysis of total RNA, lanes 1, 3–5 and 7–9 or oligo-dT purified RNA, lanes 2 and 6. RNA was isolated from WT (YF336), lanes 1 and 2; <i>rrp47</i>Δ (YF1465), lane 3; WT (YF1444), lane 4; <i>rrp6</i>Δ (YSB2244), lanes 5 and 6; WT (YF1977), lane 7; <i>dis3</i> (D171A) (YF1978), lane 8 and <i>dis3</i> (D551N) (YF1979), lane 9. RNA was resolved on a 1.2% agarose gel and probed for <i>TLC1</i> RNA. 25 and 18S rRNAs are shown below. Numbers below indicate fold increase in poly(A)+, poly(A) − <i>TLC1</i> RNA levels relative to WT and poly(A)+/poly(A) − ratio. D) Accumulation of poly(A)+ <i>TER1</i> RNA in <i>S. pombe</i> exosome mutant strains. Northern blot analysis of total RNA, lanes 1, 3 and 5, or oligo-dT purified RNA, lanes 2, 4 and 6 from WT (YP34), lanes 1 and 2; <i>rrp6</i>Δ (YP35), lanes 3 and 4; <i>dis3-54</i> (YP50), lanes 5 and 6. RNA was resolved on a 5% PAGE and <i>TER1</i> RNA was visualized with the probe depicted in (A). Positions of poly(A)+ and poly(A) − <i>TER1</i> RNAs are indicated with grey boxes. 18S rRNA is shown below. Numbers below indicate fold increase in RNA levels relative to WT.</p