The exoribonuclease Dis3L2 defines a novel eukaryotic RNA degradation pathway
2013; Springer Nature; Volume: 32; Issue: 13 Linguagem: Inglês
10.1038/emboj.2013.63
ISSN1460-2075
AutoresMichał Małecki, Sandra C. Viegas, Tiago Carneiro, Paweł Golik, Clémentine Dressaire, Miguel Godinho Ferreira, Cecília M. Arraiano,
Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle15 March 2013free access Source Data The exoribonuclease Dis3L2 defines a novel eukaryotic RNA degradation pathway Michal Malecki Michal Malecki Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland Search for more papers by this author Sandra C Viegas Sandra C Viegas Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal Search for more papers by this author Tiago Carneiro Tiago Carneiro Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Pawel Golik Pawel Golik Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland Search for more papers by this author Clémentine Dressaire Clémentine Dressaire Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal Search for more papers by this author Miguel G Ferreira Miguel G Ferreira Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Cecília M Arraiano Corresponding Author Cecília M Arraiano Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal Search for more papers by this author Michal Malecki Michal Malecki Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland Search for more papers by this author Sandra C Viegas Sandra C Viegas Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal Search for more papers by this author Tiago Carneiro Tiago Carneiro Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Pawel Golik Pawel Golik Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland Search for more papers by this author Clémentine Dressaire Clémentine Dressaire Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal Search for more papers by this author Miguel G Ferreira Miguel G Ferreira Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Cecília M Arraiano Corresponding Author Cecília M Arraiano Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal Search for more papers by this author Author Information Michal Malecki1,2, Sandra C Viegas1,‡, Tiago Carneiro3,‡, Pawel Golik2, Clémentine Dressaire1, Miguel G Ferreira3 and Cecília M Arraiano 1 1Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal 2Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland 3Instituto Gulbenkian de Ciência, Oeiras, Portugal ‡These authors contributed equally to this work. *Corresponding author. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal. Tel.:+351 214469547; Fax:+351 214469549; E-mail: [email protected] The EMBO Journal (2013)32:1842-1854https://doi.org/10.1038/emboj.2013.63 There is a Have you seen? (July 2013) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The final step of cytoplasmic mRNA degradation proceeds in either a 5′-3′ direction catalysed by Xrn1 or in a 3′-5′ direction catalysed by the exosome. Dis3/Rrp44, an RNase II family protein, is the catalytic subunit of the exosome. In humans, there are three paralogues of this enzyme: DIS3, DIS3L, and DIS3L2. In this work, we identified a novel Schizosaccharomyces pombe exonuclease belonging to the conserved family of human DIS3L2 and plant SOV. Dis3L2 does not interact with the exosome components and localizes in the cytoplasm and in cytoplasmic foci, which are docked to P-bodies. Deletion of dis3l2+ is synthetically lethal with xrn1Δ, while deletion of dis3l2+ in an lsm1Δ background results in the accumulation of transcripts and slower mRNA degradation rates. Accumulated transcripts show enhanced uridylation and in vitro Dis3L2 displays a preference for uridylated substrates. Altogether, our results suggest that in S. pombe, and possibly in most other eukaryotes, Dis3L2 is an important factor in mRNA degradation. Therefore, this novel 3′-5′ RNA decay pathway represents an alternative to degradation by Xrn1 and the exosome. Introduction Cytoplasmic mRNAs are degraded by different pathways to ensure a fast and efficient regulation of the transcriptome at the post-transcriptional level (Meyer et al, 2004; Garneau et al, 2007; Houseley and Tollervey, 2009; Balagopal et al, 2012; Schoenberg and Maquat, 2012). Two general pathways of mRNA degradation were identified using the S. cerevisiae model. Regardless of the mechanism triggering degradation, the final step of RNA decay proceeds in either 5′-3′ or 3′-5′ direction catalysed by Xrn1 exoribonuclease or the exosome complex, respectively. Deadenylation is usually the first step in both pathways. After deadenylation, the mRNA cap structure can be removed by the Dcp1–Dcp2 decapping complex. The decapping step is activated by a subset of factors that include the Lsm1-7 complex (Nissan et al, 2010). After cap removal, mRNA is accessible to the Xrn1 exonuclease which can then digest it in the 5′-3′ direction. Decapped or deadenylated mRNAs can also be degraded in the 3′-5′ direction by the exosome complex. The exosome contributes to the processing, quality control, and turnover of a large number of cellular RNAs (Mitchell et al, 1997; Houseley et al, 2006; Schaeffer et al, 2011). Exosome degradation of cytoplasmic RNAs is facilitated by the SKI complex (Anderson and Parker, 1998). In S. cerevisiae, the cytoplasmic exosome consists of nine inactive subunits (core ring structure) plus an active ribonuclease Rrp44/Dis3 (Dziembowski et al, 2007). This is a 3′-5′ hydrolytic exonuclease belonging to the ubiquitous RNase II family of enzymes. Beyond the conserved catalytic RNB domain (Frazão et al, 2006), it additionally contains a PIN domain with endonucleolytic activity (Lebreton et al, 2008; Schaeffer et al, 2009; Schneider et al, 2009). Xrn1 together with other proteins involved in decapping and 5′-3′ degradation can be localized in nucleoprotein aggregates named processing bodies (P-bodies) (Sheth and Parker, 2003; Decker and Parker, 2012). Part of the 5′-3′ mRNA degradation occurs inside these cytoplasmic structures. However messengers can also be stored inside P-bodies in an untranslational state to be later released to re-initiate translation (Brengues et al, 2005). Other aggregates found in the cytoplasm, which have a different composition from P-bodies, are the stress granules (Anderson and Kedersha, 2008; Decker and Parker, 2012). Stress granules are formed in response to stress, and can store mRNAs ready to re-initiate translation. The relationship between these two types of granules is unclear, as some components are found in both P-bodies and stress granules, and stress granules can be generated from P-bodies (Buchan et al, 2008). S. cerevisiae has been an excellent model to study cytoplasmic mRNA degradation. However, there are a substantial number of processes and enzymes conserved in eukaryotes which are absent in S. cerevisiae such as small RNA induced transcript silencing (Drinnenberg et al, 2009) and post-transcriptional modifications that can target RNAs to degradation, such as CUCU sequences (Morozov et al, 2010) or short uridine stretches (Rissland and Norbury, 2009) added to 3′-ends of mRNAs. Recent findings show that the composition of the exosome complex differs between S. cerevisiae and humans. In budding yeast, Rrp44/Dis3 localizes in the nucleus and cytoplasm and interacts with the 9-subunit exosome ring (Synowsky et al, 2009). In human genome, there are three Dis3 isoforms: DIS3, DIS3L, and DIS3L2. DIS3 is nuclear, and DIS3L and DIS3L2 are cytoplasmic (Staals et al, 2010; Tomecki et al, 2010; Astuti et al, 2012). DIS3 and DIS3L were shown to interact with the exosome ring; however, it is not clear whether DIS3L2 interacts with the exosome. DIS3L2 has a conserved catalytic RNB domain but the N-terminal PIN and CR3 domains, which facilitate interaction with the exosome (Schaeffer et al, 2012; Makino et al, 2013), are not conserved. Mutations in the human DIS3L2 gene were found in individuals with Perlman syndrome—a congenital overgrowth disease. At the molecular level, DIS3L2 depletion was shown to cause mitotic abnormalities due to the deregulation of the expression of mitotic control proteins (Astuti et al, 2012). Different isoforms of Dis3 protein seem to be a conserved feature among eukaryotes. In Arabidopsis thaliana, a protein called SOV, which has a conserved RNB domain, was suggested to be involved in cytoplasmic mRNA metabolism (Zhang et al, 2010). Although there are no Dis3L2 isoforms in S. cerevisiae, we have identified the Dis3L2 homologue in S. pombe. Fission yeast seems to share more conservation in RNA metabolism with higher eukaryotes than budding yeast. For instance, RNA interference (Moazed, 2009) or transcript uridylation, which is an evolutionary conserved mechanism of gene regulation (Schmidt and Norbury, 2010; Choi et al, 2012). Therefore, S. pombe appears to be a promising model system to investigate the cellular role of Dis3L2. Our phylogenetic analyses proved that the novel identified S. pombe nuclease belongs to the same protein family as A. thaliana SOV and human Dis3L2, and that members of this family are conserved throughout eukaryotes. Our results show that S. pombe Dis3L2 is involved in cytoplasmic mRNA degradation, and its function is independent of the exosome. This indicates that in S. pombe and possibly in most other eukaryotes, cytoplasmic mRNAs can be degraded in the 3′-5′ direction not only by the exosome complex, but alternatively by the Dis3L2 exonuclease. This discovery brings additional complexity to the current models of cytoplasmic RNA degradation. Results Identification of a new exoribonuclease in S. pombe Using S. cerevisiae Dis3p/Rrp44p sequence as a query, five genes encoding proteins with RNB domain were identified in the S. pombe genome (Supplementary Figure S1). SPBC26H8 (dis3+) gene product is a homologue of S. cerevisiae Dis3p, the active component of the exosome complex involved in RNA metabolism in both cytoplasm and nucleus (Dziembowski et al, 2007). SPCC23B6.06 gene encodes the Par1 protein, a homologue of S. cerevisiae Dss1p exonuclease crucial for RNA processing and degradation in mitochondria (Dziembowski et al, 2003; Malecki et al, 2007). A similar mitochondrial function was reported for Par1 (Hoffmann et al, 2008). SPCC16C4.09 gene encodes Sts5, homologue of S. cerevisiae Ssd1p, a protein reported to be a translation repressor involved in cell wall biogenesis and polar growth (Kurischko et al, 2011). The phenotype of fission yeast sts5+ deletion suggests a similar function for this protein (Toda et al, 1996). The other two genes identified, SPAC2C4.07C and SPBC609.01, do not have obvious homologues in S. cerevisiae genome, and their functions have not previously been investigated in S. pombe. To distinguish which of the identified genes can be an active exoribonuclease, we have compared the sequences of the active sites of their RNB domains using Clustal alignment (Figure 1A) (Thompson et al, 1997). The sequence of three conserved aspartic acids in the active site of the RNB-like enzymes is essential for exonucleolytic activity (Frazão et al, 2006). Alignment results confirmed that both S. pombe proteins, Dis3 and Par1, share conservation of the active site consensus. From the other sequences analysed, only SPAC2C4.07c retained the conserved architecture of the active site, making it a candidate for an active exoribonuclease. Figure 1.Fission yeast gene SPAC2C4.07c encodes an active RNase II family exonuclease with no homologue in S. cerevisiae. (A) S. pombe genome encodes five proteins with RNB domain. Sequence alignment depicts exonucleolytic active site of RNB domain of fission yeast proteins, compared with S. cerevisiae Rrp44/Dis3 sequence. Conserved aspartic acid in the central part of the active site is marked with the star. (B) Product of SPAC2C4.07c gene is an active exonuclease in vitro. Around 0.5 pmol of the wild-type protein product (WT) and the mutated version (D461N) were incubated with 2 pmol of the 20-nt 5′-radioactively labelled RNA substrate 1 (see below). After indicated times (T min), reactions were stopped and products separated on a denaturing polyacrylamide gel. The same substrate was incubated with 0.1 U of RNase ONE (Promega). Migration of the reaction substrates and products is indicated. (C) Product of SPAC2C4.07c gene is able to digest RNA in the context of secondary structures. Around 0.5 pmol of WT and mutated version (D461N) was incubated with 0.2 pmol of single-stranded substrate 2 (ss) or substrate 3 (ds), which is substrate 2 annealed with DNA oligonucleotide (see below). After the indicated times, reactions were stopped and products were separated on a denaturing polyacrylamide gel. Upper panel depicts schematic representation of the RNA substrates used for the reaction (left) and the control of the annealing of the double-stranded substrate (right) on a native polyacrylamide gel. RNA substrate 1: (GUUUUGUAUAGAAAUCAAUG); RNA substrate 2: (CCCGACACCAACCACUAAAAAAAAAAAAAA); substrate 3 (hybrid DNA/RNA): (DNA oligonucleotide: AGTGGTTGGTGTCGGG/RNA oligonucleotide: substrate 2). Download figure Download PowerPoint To confirm the exoribonucleolytic activity of SPAC2C4.07c gene product, a mutant was constructed having a single amino-acid substitution of aspartic acid to asparagine (D461N) and both protein versions were purified (Supplementary Figure S2). This substitution was reported to completely abolish the exonucleolytic activity of the enzymes of RNase II family (Amblar and Arraiano, 2005; Frazão et al, 2006). The wild-type protein digested a 5′-end labelled single-stranded RNA leaving a 3-nt 5′-labelled end product and we could not observe any reaction by-product, suggesting that degradation is due to a 3′-5′ processive exonucleolytic activity characteristic of the RNase II family (Figure 1B). Similarly, any reaction by-products were observed when an internally labelled RNA was used as a substrate, and uridine mononucleotides were detected as the main reaction products confirming processivity of the exonucleolytic reaction (Supplementary Figure S3). The wild-type protein also digested the RNA strand of a double-stranded RNA/DNA substrate having a 3′ RNA overhang (Matos et al, 2012) (Figure 1C). Our results prove that the product of SPAC2C4.07c is an active exonuclease in vitro and can degrade RNA even in the context of secondary structures. The mutated protein version (D461N) was not active (Figure 1B and C), confirming that the activity observed was specific of the SPAC2C4.07c gene product. SPAC2C4.07c gene product belongs to conserved Dis3L2 family of exonucleases We have performed phylogenetic analysis in order to place the newly discovered S. pombe RNase in relation to the known eukaryotic proteins and to investigate conservation of Dis3 isoforms. Genomic sequences available for eukaryotic species representing different branches of the tree of life were analysed using BLASTn to find encoded proteins similar to the Dis3p of budding yeast. The sequences corresponding to mitochondrial exonucleases were discarded. Final collection of sequences (summarized in Supplementary Table 3) was subjected to phylogenetic analysis using the Neighbour Joining (NJ), Maximum Parsimony (MP), and Bayesian inference (BI) methods. The obtained trees are shown in Figure 2A (NJ) and Supplementary Figure S4 (MP and BI). Figure 2.Phylogenetic comparison of different Dis3 homologues in eukaryotes reveals that Dis3-like proteins can be divided into three distinct groups, corresponding to Dis3, Dis3L, and Dis3L2. Dis3 and Dis3L2 are found almost universally in Metazoa, plants, and Fungi, while proteins from the Dis3L group are found only in vertebrates and in a single invertebrate species (Nematostella vectensis). (A) A neighbour joining tree (BioNJ) of 43 eukaryotic Dis3-like proteins. Numbers correspond to percentage bootstrap support (1000 replicates) for nodes. Three main groups, corresponding to Dis3, Dis3L, and Dis3L2 proteins have 100% support, and the SPAC2C4.07c gene product of S. pombe belongs to the Dis3L2 group. (B) Clustering of the Dis3-like proteins (same set as in A) on the basis of pairwise BLAST similarity using CLANS also reveals three distinct groups of Dis3, Dis3L, and Dis3L2 sequences, with the Dis3 group forming the tightest cluster. Again, the SPAC2C4.07c gene product of S. pombe belongs to the Dis3L2 group. Download figure Download PowerPoint Regardless of the phylogenetic inference method used, the resulting trees show the same general topology with significant support. The Dis3 homologues fall into three main branches, corresponding to the Dis3, Dis3L, and Dis3L2 proteins. Dis3 and Dis3L2 are found in all eukaryotic clades—metazoans, plants, and some fungi. A few fungal species (including S. cerevisiae) do not have Dis3L and Dis3L2, containing only one sequence of this family belonging to the Dis3 group. The presence of the third (Dis3L) isoform is restricted to metazoans and, in most cases, to vertebrates, with the intriguing exception of Nematostella vectensis (starlet sea anemone)—the only known invertebrate possessing all three isoforms of Dis3. The three groups are most likely the product of two ancient gene duplications that occurred very early during evolution. In all phylogenetic trees (Figure 2A; Supplementary Figure S4), the SPAC2C4.07c gene product consistently clusters with the Dis3L2 proteins (including human DIS3L2 and SOV of A. thaliana), and will be henceforth called the Dis3L2 of S. pombe. Therefore, the fission yeast genome encodes two Dis3 isoforms, one from the Dis3 group and other from the Dis3L2 group. We also performed the CLANS (CLuster ANalysis of Sequences) analysis, which clusters the sequences on the basis of pairwise BLAST scores. The results confirm the clear division of three Dis3-like protein families in eukaryotes, with the Dis3 group being the most conserved, thus tightly connected, and the Dis3L and Dis3L2 groups being more divergent (Figure 2B). Again, in this analysis the Dis3L2 of S. pombe clearly clusters with Dis3L2 group that includes human DIS3L2. Interestingly, alignments of Dis3 homologues from different species unrevealed peculiar active site sequence conservation: all Dis3 homologues have isoleucine between the three conserved aspartic acids in the active site (DIDD), all Dis3L homologues have valine in the same site (DVDD), and all Dis3L2 have leucine (DLDD) in that site (Supplementary Figure S4C). Dis3L2 protein localizes in the cytoplasm and foci mostly docked to P-bodies We have fused GFP to the 3′-end of S. pombe dis3l2+ in its genomic locus and analysed the localization of the fusion protein. Dis3L2-GFP, when expressed from its endogenous promoter, has a slightly different localization in the cell than when overexpressed (Matsuyama et al, 2006). The protein localizes in the cytoplasm and gives a general signal equally distributed throughout the cytosol; nevertheless, we could observe additional foci of higher fluorescence (Figure 3A; Supplementary Figure S5). The most widely studied cytoplasmic aggregates involved in mRNA metabolism are P-bodies and stress granules and we decided to check if Dis3L2 co-localized with any of these structures. Figure 3.Dis3L2 co-localizes with P-bodies. (A) Dis3L2 localizes in the cytoplasm and cytoplasmic foci. Cells expressing Dis3L2-GFP were grown to mid-log phase in minimal medium (EMM) and the localization of epitope-tagged protein was determined by fluorescence microscopy. (B, C) Dis3L2-GFP was examined for co-localization with Dcp2-RFP (B) or PabP-RFP (C). Cells expressing Dis3L2-GFP and Dcp2-RFP were grown to mid-log phase in minimal medium (EMM) and either immediately observed in the microscope (glucose), or deprived for glucose for 10 min and subsequently examined (no glucose) (B). Similar experiment was performed for the comparison of cells expressing Dis3L2-GFP and PabP-RFP (C) except that fission yeast was grown on full media (YES) (see Results). Examples of Dis3L2 aggregates docked to P-bodies in (B) were marked with circles. Scale bars represent 5 μm. Download figure Download PowerPoint To check Dis3L2 co-localization with P-bodies, we used the P-body marker Dcp2 that is an active subunit of the RNA decapping complex (Ling et al, 2011). Yeast strains with Dis3L2 and Dcp2 fused with different fluorescent markers were constructed. Strains were grown in the minimal media (EMM) up to the early exponential growth phase and immediately subjected to microscopy analysis. In these conditions, most of the Dis3L2-GFP foci appear to be adjacent to Dcp2-RFP labelled P-bodies (Figure 3B; Supplementary Figure S5). In some cases, complete co-localization could also be observed. There are also a few weaker Dis3L2-GFP foci that do not co-localize with Dcp2-RFP, while the opposite situation barely happens. However, the co-localization pattern differs when we incubate the cells in glucose deprived media prior to microscopy. These conditions are known to increase the number and size of P-bodies in both S. cerevisiae and S. pombe (Teixeira et al, 2005; Nilsson and Sunnerhagen, 2011). Interestingly, after glucose deprivation the Dis3L2 and Dcp2 signals completely co-localize (Figure 3B). It was shown for yeast that stress granules can be docked to the P-bodies in stress conditions (Buchan et al, 2008; Nilsson and Sunnerhagen, 2011). We checked the co-localization of Dis3L2 with the stress granule marker Pabp (poly(A) binding protein) (Nilsson and Sunnerhagen, 2011). Interestingly, the strain containing both fusion proteins could not grow on minimal media (data not shown). In cells grown in the rich media (YES), only a few weak stress granules could be detected (Figure 3C). The observed granules were docked or co-localized to the Dis3L2-GFP signal. However, a substantial number of Dis3L2-GFP foci did not co-localize to the stress granules. A similar localization pattern was observed in glucose deprived cells (Figure 3C). Our results suggest that Dis3L2 can either dock or co-localize to P-bodies, as we observe a substantial number of Dis3L2 foci localizing to P-bodies even in the conditions when stress granules are hardly detected. Dis3L2 fails to interact with the exosome complex In humans, both DIS3 and DIS3L interact with the exosome ring, whereas DIS3L2 was not found in exosome pull downs, suggesting a lack of interaction (Staals et al, 2010; Tomecki et al, 2010). To check if the S. pombe Dis3L2 protein can interact with the exosome complex, we first investigated localization of the exosome ring protein Rrp43 and the active subunit Dis3. Similar to S. cerevisiae, the exosome complex is localized mainly in the nucleus (Figure 4A) (Huh et al, 2003; Yamanaka et al, 2010). The signal for both exosome proteins in cytoplasm was hardly distinguishable from the background. Co-localization of Dis3L2-GFP and Dis3-RFP showed that Dis3-RFP is mostly located in nucleus and Dis3L2-GFP in the cytoplasm. The results suggest that the exosome complex does not interact with Dis3L2 since they have different spatial distribution. However, similarly to the situation in S. cerevisiae, there is probably a small fraction of the exosome complex present in the cytosol. Figure 4.Dis3L2 fails to interact with the exosome complex. (A) Dis3L2 does not co-localize with the exosome complex components. Cells expressing Dis3L2-GFP, Rrp43-GFP, Dis3-GFP or both Dis3-RFP and Dis3L2-GFP were grown in the minimal media (EMM) to mid-log phase and protein localization determined by fluorescence microscopy. Scale bars represent 5 μm. (B) Dis3L2 does not co-purify with the exosome complex components. Cells expressing exosome complex components (Dis3 or Rrp43) fused with TAP tag sequence were grown to mid-log phase in rich media, subsequently tagged proteins were purified according to standard protocol (Rigaut et al, 1999). Part of each elution from the calmodulin resin was separated on SDS–PAGE gel and silver stained (gel on the left), the remaining part was subjected to mass spectrometry analysis. Table presented on the right lists all known exosome subunits and interacting proteins identified in the elutions from Dis3-TAP and Rrp43-TAP. Download figure Download PowerPoint Since our microscopy data could not exclude the possibility that cytoplasmic fraction of the exosome interacted with Dis3L2, we decided to use Tandem Affinity Purification (TAP) (Rigaut et al, 1999) coupled to mass spectrometry analysis to identify proteins interacting with Dis3L2, Dis3 and the exosome ring. TAP tag sequence was fused with 3′-ends of dis3l2+, dis3+ and rrp43+ under their endogenous loci. Due to the difficulties with Dis3L2-TAP purification, we proceeded with the analysis of the TAP fusions of the exosome components—Rrp43 and Dis3. In both cases, after purification and analysis on silver stained protein gels, we could identify all the main exosome complex components co-purifying with the tagged proteins (Figure 4B). Parts of the fractions from the final elution were subjected to mass spectrometry analysis to identify all the co-purified proteins. As expected, we could identify all the nine main exosome subunits co-purifying with both the Rrp43-TAP and the Dis3-TAP proteins. Moreover, we identified additional proteins that were reported to interact with the exosome complex, either in the nucleus (Rrp47, Mpp6, and Rpp6) or in the cytoplasm (Ski7) (Synowsky et al, 2009; Marshall et al, 2013). Dis3L2 was not detected among the proteins identified in either sample, suggesting that this protein acts independently from the exosome complex (list of all identified proteins in Supplementary Table 4). The homologue of S. cerevisiae cytoplasmic exosome cofactor Ski7 co-purified together with both Dis3-TAP and exosome ring protein Rrp43-TAP indicating that, in S. pombe, the same 10 subunit exosome complex may act both in the cytoplasm and in the nucleus. Dis3L2+ genetically interacts with components of cytoplasmic mRNA degradation pathway Dis3L2 enzymatic activity, and the fact that it partially co-localizes with P-bodies, suggests a role in cytoplasmic mRNA degradation. We checked the genetic interactions between dis3l2+ and different components of mRNA degradation pathway by constructing double deletion strains. Genes chosen for the deletion together with dis3l2+ were xrn1+ encoding the exonuclease that drives degradation from the 5′-ends, ski2+ encoding a component of the Ski complex that connects the exosome to its cytoplasmic substrates, and lsm1+ encoding a protein that, in complex with six other Lsm proteins and Pat1, binds to the 3′-end of the transcripts protecting them from 3′ trimming, activating decapping and 5′-3′ degradation (He and Parker, 2001). The Lsm complex is also involved in the P-body formation (Teixeira and Parker, 2007). Tetrad analysis from crosses of single mutants showed that dis3l2Δ is synthetically lethal with xrn1Δ (Figure 5A). In S. cerevisiae XRN1 gene deletion is synthetically lethal with deletion of SKI complex components, crucial for 3′-5′ mRNA degradation by the exosome (Anderson and Parker, 1998). Lethality of double XRN1 SKI deletion strains is thought to be due to a blockage of both 5′-3′ and 3′-5′ RNA degradation pathways. Similarly, synthetic lethality of dis3l2Δ with xrn1Δ, together with the observed Dis3L2 3′-5′ exonucleolytic activity, suggests that dis3l2+ deletion impairs 3′-5′ mRNA degradation pathway. Lethality of double mutant suggests that cytoplasmic exosome cannot compensate for Dis3L2 function. Similarly, in the xrn1Δ strain Dis3L2 cannot fully compensate for loss of cytoplasmic exosome function since xrn1Δski2Δ mutant is inviable (Supplementary Figure S6). Figure 5.Genetic interactions between dis3l2+ and the components of cytoplasmic RNA degradation pathway. (A) dis3l2+ deletion is synthetically lethal with deletion of xrn1+. Haploid dis3l2+::kan cells were crossed with xrn1+::hph strain. Resulting diploids were sporulated and tetrads were dissected on YES plates. In the bottom table, the genotypes of the germinated spores are described: WT—wild-type strain, X—xrn1Δ, D—dis3l2Δ. Genotypes of the spores were analysed by their ability to grow on the selective media and by colony PCR. (B) dis3l2+ deletion enhances the growth defect of lsm1Δ strain. Overnight yeast cultures were diluted in the fresh media (EMM) to OD600 of 0.1, subsequently grown at 32°C and the OD monitored over time. Download figure Download PowerPoint Single dis3l2+ deletion did not have much impact on S. pombe cells growth in the conditions tested (Figure 5B). Deletion of ski2+ or double deletion of dis3l2+ together with ski2+ also did not have significant effect on growth (Figure 5B). Likewise, deletions of SKI genes in S. cerevisiae have only mild effect on growth, which is due to the redundancy of the 5′ and 3′ direction degradation pathway
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