Selective termination of lnc RNA transcription promotes heterochromatin silencing and cell differentiation
2017; Springer Nature; Volume: 36; Issue: 17 Linguagem: Inglês
10.15252/embj.201796571
ISSN1460-2075
AutoresLeila Touat‐Todeschini, Yuichi Shichino, Mathieu Dangin, Nicolas Thierry‐Mieg, Benoît Gilquin, Edwige Hiriart, Ravi Sachidanandam, Emeline Lambert, Janine Brettschneider, M. Reuter, Jan Kadlec, Ramesh S. Pillai, Akira Yamashita, Masayuki Yamamoto, André Verdel,
Tópico(s)Plant Virus Research Studies
ResumoArticle1 August 2017free access Source DataTransparent process Selective termination of lncRNA transcription promotes heterochromatin silencing and cell differentiation Leila Touat-Todeschini Institut for Advanced Biosciences, UMR InsermU1209/CNRS5309/UGA, University of Grenoble Alpes, Grenoble, France Search for more papers by this author Yuichi Shichino orcid.org/0000-0002-0093-1185 Laboratory of Cell Responses, National Institute for Basic Biology, Okazaki, Aichi, Japan Search for more papers by this author Mathieu Dangin Institut for Advanced Biosciences, UMR InsermU1209/CNRS5309/UGA, University of Grenoble Alpes, Grenoble, France Search for more papers by this author Nicolas Thierry-Mieg TIMC-IMAG, University of Grenoble Alpes, Grenoble, France CNRS, TIMC-IMAG, UMR CNRS 5525, Grenoble, France Search for more papers by this author Benoit Gilquin CEA, LETI, CLINATEC, MINATEC Campus, University of Grenoble Alpes, Grenoble, France Search for more papers by this author Edwige Hiriart Institut for Advanced Biosciences, UMR InsermU1209/CNRS5309/UGA, University of Grenoble Alpes, Grenoble, France Search for more papers by this author Ravi Sachidanandam orcid.org/0000-0001-9844-4459 Department of Oncological Sciences, Icahn School of Medicine at Sinai, New York, NY, USA Search for more papers by this author Emeline Lambert Institut for Advanced Biosciences, UMR InsermU1209/CNRS5309/UGA, University of Grenoble Alpes, Grenoble, France Search for more papers by this author Janine Brettschneider European Molecular Biology Laboratory, Grenoble Outstation, University of Grenoble Alpes-EMBL-CNRS, Grenoble, France Unit for Virus Host-Cell Interactions, University of Grenoble Alpes-EMBL-CNRS, Grenoble, France Search for more papers by this author Michael Reuter European Molecular Biology Laboratory, Grenoble Outstation, University of Grenoble Alpes-EMBL-CNRS, Grenoble, France Unit for Virus Host-Cell Interactions, University of Grenoble Alpes-EMBL-CNRS, Grenoble, France Search for more papers by this author Jan Kadlec European Molecular Biology Laboratory, Grenoble Outstation, University of Grenoble Alpes-EMBL-CNRS, Grenoble, France Unit for Virus Host-Cell Interactions, University of Grenoble Alpes-EMBL-CNRS, Grenoble, France Institut de Biologie Structurale (IBS), CEA, CNRS, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Ramesh Pillai Institut de Biologie Structurale (IBS), CEA, CNRS, Université Grenoble Alpes, Grenoble, France Department of Molecular Biology, University of Geneva, Geneva 4, Switzerland Search for more papers by this author Akira Yamashita Laboratory of Cell Responses, National Institute for Basic Biology, Okazaki, Aichi, Japan Department of Basic Biology, School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Aichi, Japan Search for more papers by this author Masayuki Yamamoto Laboratory of Cell Responses, National Institute for Basic Biology, Okazaki, Aichi, Japan Department of Basic Biology, School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Aichi, Japan Search for more papers by this author André Verdel Corresponding Author [email protected] orcid.org/0000-0001-6048-3794 Institut for Advanced Biosciences, UMR InsermU1209/CNRS5309/UGA, University of Grenoble Alpes, Grenoble, France Search for more papers by this author Leila Touat-Todeschini Institut for Advanced Biosciences, UMR InsermU1209/CNRS5309/UGA, University of Grenoble Alpes, Grenoble, France Search for more papers by this author Yuichi Shichino orcid.org/0000-0002-0093-1185 Laboratory of Cell Responses, National Institute for Basic Biology, Okazaki, Aichi, Japan Search for more papers by this author Mathieu Dangin Institut for Advanced Biosciences, UMR InsermU1209/CNRS5309/UGA, University of Grenoble Alpes, Grenoble, France Search for more papers by this author Nicolas Thierry-Mieg TIMC-IMAG, University of Grenoble Alpes, Grenoble, France CNRS, TIMC-IMAG, UMR CNRS 5525, Grenoble, France Search for more papers by this author Benoit Gilquin CEA, LETI, CLINATEC, MINATEC Campus, University of Grenoble Alpes, Grenoble, France Search for more papers by this author Edwige Hiriart Institut for Advanced Biosciences, UMR InsermU1209/CNRS5309/UGA, University of Grenoble Alpes, Grenoble, France Search for more papers by this author Ravi Sachidanandam orcid.org/0000-0001-9844-4459 Department of Oncological Sciences, Icahn School of Medicine at Sinai, New York, NY, USA Search for more papers by this author Emeline Lambert Institut for Advanced Biosciences, UMR InsermU1209/CNRS5309/UGA, University of Grenoble Alpes, Grenoble, France Search for more papers by this author Janine Brettschneider European Molecular Biology Laboratory, Grenoble Outstation, University of Grenoble Alpes-EMBL-CNRS, Grenoble, France Unit for Virus Host-Cell Interactions, University of Grenoble Alpes-EMBL-CNRS, Grenoble, France Search for more papers by this author Michael Reuter European Molecular Biology Laboratory, Grenoble Outstation, University of Grenoble Alpes-EMBL-CNRS, Grenoble, France Unit for Virus Host-Cell Interactions, University of Grenoble Alpes-EMBL-CNRS, Grenoble, France Search for more papers by this author Jan Kadlec European Molecular Biology Laboratory, Grenoble Outstation, University of Grenoble Alpes-EMBL-CNRS, Grenoble, France Unit for Virus Host-Cell Interactions, University of Grenoble Alpes-EMBL-CNRS, Grenoble, France Institut de Biologie Structurale (IBS), CEA, CNRS, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Ramesh Pillai Institut de Biologie Structurale (IBS), CEA, CNRS, Université Grenoble Alpes, Grenoble, France Department of Molecular Biology, University of Geneva, Geneva 4, Switzerland Search for more papers by this author Akira Yamashita Laboratory of Cell Responses, National Institute for Basic Biology, Okazaki, Aichi, Japan Department of Basic Biology, School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Aichi, Japan Search for more papers by this author Masayuki Yamamoto Laboratory of Cell Responses, National Institute for Basic Biology, Okazaki, Aichi, Japan Department of Basic Biology, School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Aichi, Japan Search for more papers by this author André Verdel Corresponding Author [email protected] orcid.org/0000-0001-6048-3794 Institut for Advanced Biosciences, UMR InsermU1209/CNRS5309/UGA, University of Grenoble Alpes, Grenoble, France Search for more papers by this author Author Information Leila Touat-Todeschini1, Yuichi Shichino2,‡, Mathieu Dangin1,‡, Nicolas Thierry-Mieg3,4, Benoit Gilquin5, Edwige Hiriart1, Ravi Sachidanandam6, Emeline Lambert1, Janine Brettschneider7,8, Michael Reuter7,8, Jan Kadlec7,8,9, Ramesh Pillai9,10, Akira Yamashita2,11, Masayuki Yamamoto2,11 and André Verdel *,1 1Institut for Advanced Biosciences, UMR InsermU1209/CNRS5309/UGA, University of Grenoble Alpes, Grenoble, France 2Laboratory of Cell Responses, National Institute for Basic Biology, Okazaki, Aichi, Japan 3TIMC-IMAG, University of Grenoble Alpes, Grenoble, France 4CNRS, TIMC-IMAG, UMR CNRS 5525, Grenoble, France 5CEA, LETI, CLINATEC, MINATEC Campus, University of Grenoble Alpes, Grenoble, France 6Department of Oncological Sciences, Icahn School of Medicine at Sinai, New York, NY, USA 7European Molecular Biology Laboratory, Grenoble Outstation, University of Grenoble Alpes-EMBL-CNRS, Grenoble, France 8Unit for Virus Host-Cell Interactions, University of Grenoble Alpes-EMBL-CNRS, Grenoble, France 9Institut de Biologie Structurale (IBS), CEA, CNRS, Université Grenoble Alpes, Grenoble, France 10Department of Molecular Biology, University of Geneva, Geneva 4, Switzerland 11Department of Basic Biology, School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Aichi, Japan ‡These authors contributed equally to this work *Corresponding author. Tel: +33 476 549 422; E-mail: [email protected] EMBO J (2017)36:2626-2641https://doi.org/10.15252/embj.201796571 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 Abstract Long non-coding RNAs (lncRNAs) regulating gene expression at the chromatin level are widespread among eukaryotes. However, their functions and the mechanisms by which they act are not fully understood. Here, we identify new fission yeast regulatory lncRNAs that are targeted, at their site of transcription, by the YTH domain of the RNA-binding protein Mmi1 and degraded by the nuclear exosome. We uncover that one of them, nam1, regulates entry into sexual differentiation. Importantly, we demonstrate that Mmi1 binding to this lncRNA not only triggers its degradation but also mediates its transcription termination, thus preventing lncRNA transcription from invading and repressing the downstream gene encoding a mitogen-activated protein kinase kinase kinase (MAPKKK) essential to sexual differentiation. In addition, we show that Mmi1-mediated termination of lncRNA transcription also takes place at pericentromeric regions where it contributes to heterochromatin gene silencing together with RNA interference (RNAi). These findings reveal an important role for selective termination of lncRNA transcription in both euchromatic and heterochromatic lncRNA-based gene silencing processes. Synopsis The identification of new lncRNAs in fission yeast reveals a role for RNA-binding protein Mmi1 in controlling sexual differentiation via lncRNA binding, and shows how faulty transcription termination of lncRNAs can regulate the expression of neighbouring genes. Binding of the Mmi1 YTH domain to the unique lncRNA nam1 controls cell differentiation in fission yeast. Mmi1 recruitment to the nascent nam1 lncRNA promotes transcription termination at the nam1 locus. Transcription read-through at the nam1 locus silences the adjacent MAPKKK gene, a key differentiation regulator. Mmi1 also promotes termination of lncRNA transcription within heterochromatin, thus contributing to gene silencing. Introduction Long non-coding RNAs (lncRNAs) are widespread regulators of gene transcription and chromatin modification among eukaryotes. LncRNAs control gene expression, in cis or in trans, by serving as decoy or scaffold that interact with chromatin modifiers and remodelers to regulate the chromatin state of specific genomic sites (Rinn & Chang, 2012; Morris & Mattick, 2014). Studies on nuclear RNA interference (RNAi)-mediated gene silencing have further provided evidence that RNAi co-transcriptionally eliminates regulatory lncRNAs and, in addition, mediates the function of these lncRNAs in forming heterochromatin or silent chromatin in the fission yeast Schizosaccharomyces pombe and other eukaryotes (Castel & Martienssen, 2013). However, most of the co-transcriptional activity eliminating lncRNAs relies on the conserved exosome complex instead of RNAi (Kilchert et al, 2016) and, in this case, the functional and physical connections between exosome-dependent co-transcriptional elimination of lncRNAs and lncRNA-based gene silencing processes are poorly characterized. Among the extensively studied cases of regulatory lncRNAs co-transcriptionally controlled by RNAi are the S. pombe pericentromeric lncRNAs, which play a central role in the formation of heterochromatin (Buhler & Moazed, 2007; Cam et al, 2009), characterized by the methylation of histone H3 on lysine 9 (H3K9me; Grewal & Jia, 2007). Schizosaccharomyces pombe pericentromeric regions are mainly composed of DNA repeats, named dg and dh, transcribed by the RNA polymerase II (RNAPII; Djupedal et al, 2005; Kato et al, 2005). Production of dg and dh sense and anti-sense lncRNAs is believed to lead to the formation of double-stranded RNAs (dsRNAs; Reinhart & Bartel, 2002). The RNAi protein Dicer (Dcr1) processes dsRNAs into small interfering RNAs (siRNAs) that load on the RNA-induced transcriptional gene silencing (RITS) complex (Verdel et al, 2004). RITS uses the siRNAs as guides to co-transcriptionally base-pair with nascent and complementary lncRNAs (Motamedi et al, 2004; Buhler et al, 2006) that are then eliminated by a cis-acting positive feedback loop. In this loop, the targeted nascent lncRNAs serve as matrixes to locally synthesize double-stranded RNAs and produce more siRNAs and RITS that bind the nascent pericentromeric lncRNAs (Motamedi et al, 2004; Colmenares et al, 2007). In complement to RNAi, the exosome complex and its cofactor, the TRAMP complex, also contribute to the elimination of dg and dh lncRNAs (Buhler et al, 2007; Wang et al, 2008; Reyes-Turcu et al, 2011). The exosome is a highly conserved nucleocytoplasmic complex that degrades RNA (Chlebowski et al, 2013; Kilchert et al, 2016), including the nascent lncRNAs issued from pervasive transcription (Jensen et al, 2013). Because both Rrp6, a 3′ → 5′ exonuclease only present in the nuclear form of the exosome complex, and Cid14, a subunit of the TRAMP complex, localize in the vicinity of pericentromeric heterochromatin (Keller et al, 2012; Oya et al, 2013), it has been proposed that the exosome and TRAMP reinforce heterochromatin gene silencing by a cis-acting post-transcriptional gene silencing that degrades lncRNAs produced from heterochromatin regions. Like in other eukaryotes, a major function of the S. pombe nuclear exosome is to degrade co-transcriptionally the lncRNAs produced by pervasive transcription (Zhou et al, 2015). In addition, the nuclear exosome is also part of an RNA surveillance machinery that targets meiotic pre-mRNAs in a selective manner to prevent S. pombe cells from undergoing meiosis during vegetative growth (Harigaya et al, 2006; Yamanaka et al, 2010; Hiriart et al, 2012; Zofall et al, 2012; Tashiro et al, 2013). The selective targeting of the surveillance machinery is achieved by the YTH RNA-binding domain of Mmi1 (Harigaya et al, 2006; meiotic mRNA interception protein 1), which recognizes the hexameric RNA motif UNAAAC (where N can be any nucleotide) present in several copies in these meiotic transcripts (Chen et al, 2011; Hiriart et al, 2012; Yamashita et al, 2012). In parallel, Mmi1 RNA surveillance machinery also eliminates specific nascent lncRNAs by recognizing the same hexameric motif (Hiriart et al, 2012; Ard et al, 2014; Shah et al, 2014; Chatterjee et al, 2016). Mmi1-mediated elimination of the lncRNA meiRNA regulates meiosis (Hiriart & Verdel, 2013; Yamashita et al, 2016), while its elimination of the lncRNA prt1 regulates phosphate uptake (Shah et al, 2014; Chatterjee et al, 2016). Mmi1 binding to prt1 and to meiotic pre-mRNAs triggers the recruitment of RNAi proteins (Hiriart et al, 2012; Shah et al, 2014) and the formation of facultative heterochromatin (Hiriart et al, 2012; Zofall et al, 2012; Tashiro et al, 2013; Shah et al, 2014). Mmi1 also promotes the transcription termination of its targeted meiotic and lncRNA genes (Shah et al, 2014; Chalamcharla et al, 2015). It has been proposed that Mmi1/exosome-mediated transcription termination serves to prime the lncRNA targets for degradation (Shah et al, 2014), yet this remains to be tested. More broadly, the biological significance and the direct implication of this transcription termination in Mmi1-mediated gene silencing have not been addressed. According to its primary sequence, Mmi1 belongs to the large family of YTH (YT521-B Homology) RNA-binding proteins (Stoilov et al, 2002; Zhang et al, 2010; Wang & He, 2014). The recent structures of its YTH domain confirmed its overall similarity to the other YTH domains studied (Chatterjee et al, 2016; Wang et al, 2016). However, in contrast to budding yeast and mammalian YTH domains that bind to RNA by specifically recognizing the methylated adenine (m6A) RNA modification (Li et al, 2014; Theler et al, 2014; Xu et al, 2014; Zhu et al, 2014), Mmi1 YTH domain binds to RNA by recognizing the unmethylated UNAAAC motif (Chen et al, 2011; Yamashita et al, 2012; Wang et al, 2016). On the other hand, and similarly to Mmi1, other YTH domain-containing proteins associate to both mRNAs and lncRNAs (Xu et al, 2014; Patil et al, 2016), and their association with mRNAs controls RNA decay (Wang et al, 2014) and splicing (Xiao et al, 2016), while the function of their association with lncRNAs is poorly characterized. In this study, thanks to a combination of high-throughput sequencing, computational prediction, and protein structure-driven analyses, we identify new lncRNAs targeted by the YTH domain of Mmi1 and find that the co-transcriptional binding of Mmi1 to some of these lncRNAs controls sexual differentiation and heterochromatin gene silencing. We uncover that Mmi1 binding to a unique nascent lncRNA, that we named non-coding RNA associated to Mmi1 (nam1), is sufficient to control entry into sexual differentiation. Importantly, Mmi1 binding to nam1 not only promotes the recruitment of the exosome but also imposes a robust termination of transcription of nam1. We further demonstrate that, by doing so, Mmi1 prevents nam1 read-through transcription from repressing the downstream mitogen-activated protein kinase kinase kinase (MAPKKK) essential to entry into sexual differentiation. In addition, we also uncover that Mmi1 binding to pericentromeric lncRNAs mediates heterochromatin gene silencing, in particular by promoting transcription termination. Finally, we show that Mmi1-mediated termination of lncRNA transcription may not act in parallel but rather alternate during the cell cycle with the RNAi-mediated heterochromatin gene silencing. Altogether, these findings demonstrate that the selective transcription termination of lncRNA genes mediated by the YTH domain of Mmi1 regulates lncRNA-based gene silencing processes implicated in important cellular processes such as cell differentiation and heterochromatin gene silencing. Results Extensive identification of RNAs targeted by Mmi1's YTH domain To better characterize the function of Mmi1 RNA-binding protein, we searched for the RNAs targeted by Mmi1 on a genomewide scale. We first conducted Mmi1 RNA-IPs coupled to high-throughput sequencing. Thousands of RNAs were identified in both Mmi1 and control RNA-IPs, but only 27 RNAs were enriched at least twofold in all Mmi1 RNA-IPs (Fig 1A and Appendix Table S1); 15 of the 20 previously validated mRNA targets of Mmi1 (Harigaya et al, 2006; Hiriart et al, 2012) as well as eight new mRNAs were among them. As expected, the enriched mRNAs showed almost exclusively a meiotic expression profile (Appendix Table S1) and a high density of Mmi1 binding motifs UNAAAC in comparison with the complete set of S. pombe mRNAs (Fig EV1A). Interestingly, three new lncRNAs produced from different euchromatic regions and a snoRNA were also enriched in Mmi1 RNA-IPs (Fig 1A). All three lncRNAs possess an overrepresentation of UNAAAC motifs in their sequence relative to the complete set of S. pombe-annotated non-coding RNAs (Fig EV1A), suggesting that they are also targets of Mmi1. Figure 1. Extensive identification of Mmi1 RNA targets by combining RNA-IP sequencing and computational approaches A. Box plot of the enrichment of the RNAs identified by Mmi1 RNA-IPs coupled to high-throughput sequencing. The log of the average enrichments obtained from two independent RNA-IPs is plotted. The enrichment is relative to the no antibody RNA-IPs conducted in parallel with Mmi1 RNA-IPs. The boxes represent the median and the upper and lower quartiles (25% and 75%). The whiskers extend to 1.5 times the interquartile range from the box. The mRNAs and lncRNAs enriched at least twofold in both Mmi1 RNA-IPs are shown in blue and green, respectively. Crosses represent newly identified Mmi1 targets and dots known targets. B. LncRNAs predicted to be targets of Mmi1 with a high confidence by our computational approach. C. Surface representation of Mmi1 YTH domain. Area corresponding to conserved residues (according to the alignment shown in Fig EV2A) and located at the surface are highlighted in green. Mutated residues in Mmi1 YTH domain are indicated in blue for the ones located within the aromatic cage, in purple for the ones surrounding the cage, and in red for the rest. D. RNA-IPs showing the impact of Mmi1 YTH domain point mutations on Mmi1 binding to ssm4 mRNA. The lower part shows a Western blot monitoring the protein level of WT and mutant Mmi1 proteins in the cells used for the RNA-IPs. Loading was monitored using an anti-Tub1 (tubulin) antibody. E, F. RNA-IPs showing the YTH-dependent association of Mmi1 with the lncRNAs identified in (A) and (B). Data information: Average fold enrichment is shown with error bars that indicate mean average deviations for three independent experiments for (D–F). Source data are available online for this figure. Source Data for Figure 1 [embj201796571-sup-0004-SDataFig1.JPG] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Density of Mmi1 binding sites within mRNAs and lncRNAs, and computational approach developed to predict Mmi1 RNA targets Density of Mmi1 binding motif (UNAAAC) among all the annotated mRNAs and lncRNAs versus the mRNAs and lncRNAs enriched at least twofold in Mmi1 RNA-IP Seq. Diagram of the genomewide computational approach used to predict Mmi1 targets. In step 1, a hypothetical open reading frame that has eight UNAAAC motifs in its sequence is shown as an example. The double-headed arrows indicate all possible windows containing from two to eight motifs. The thick double-headed arrows correspond to the windows yielding the minimal window size (MWS) from two to eight motifs. In step 2, each box plot represents the MWSx calculated for all of the validated targets. The boxplots were produced using the "boxplot" function in R with default parameters: the boxes represent the median and the upper and lower quartiles (25% and 75%); and the whiskers attempt to capture the extreme values, but extend to at most 1.5 times the interquartile range from the box, in which case any outliers beyond this range appear as crosses. The numbers in red represent the cutoff window size (CWS) for two to eight motifs. See the Materials and Methods for more details. Download figure Download PowerPoint We conducted in parallel a computational approach to identify Mmi1 targets. Conversely, to the RNA-IPs sequencing approach, the computational approach does not require a minimal level of expression of the target RNA to be identified. The computational approach considers the number and density of UNAAAC motifs per RNA as criteria to screen among all S. pombe-annotated mRNAs and lncRNAs (Fig EV1B). Using stringent filtering conditions (see the Materials and Methods for more details), a total of 17 mRNAs mostly expressed during meiosis (Appendix Table S2) as well as five lncRNAs were identified as high-confidence targets of Mmi1 (Fig 1B). Noticeably, the set of lncRNAs included two of the lncRNAs identified by the high-throughput RNA-IP approach, the meiRNA (Hiriart et al, 2012; Yamashita et al, 2012) and, intriguingly, two lncRNAs produced from pericentromeric regions embedded within heterochromatin, suggesting that Mmi1 binding to lncRNAs may not be restricted to euchromatic lncRNAs but also encompasses heterochromatic lncRNAs expressed at low levels. In the process of characterizing Mmi1 binding to RNA, we obtained the structure of the Mmi1 YTH domain, at 1.5 Å resolution (Figs 1C and EV2A and B, and Appendix Table S3) and, based on this structure, we made a series of point mutants that may interfere with Mmi1 binding to RNA without impacting on the structure of the domain. Wild-type and mutant Mmi1 proteins were expressed in mmi1∆ cells, and their in vivo binding to ssm4, spo5, and rec8 mRNAs, three previously validated targets of Mmi1 (Hiriart et al, 2012), was monitored. Out of the 10 mutations made, mutations R351E and R381E were found to cause a marked reduction of Mmi1 binding to the three target mRNAs without reducing the protein level of Mmi1 (Figs 1D and EV2C). In agreement with the possibility that these two mutations impact on Mmi1 YTH domain binding to RNA rather than on its structure, gel filtration experiments showed similar elution patterns for R351E, R381E, and wild-type Mmi1 YTH domains (Fig EV2D), and RNA pull-down experiments indicated that both mutations negatively impact on Mmi1 binding to RNA in vitro (Fig EV2E and F). Additionally, the analysis of the subcellular localization of Mmi1 R351E and R381E proteins by immunofluorescence showed that their localization is similar to the wild-type Mmi1 protein (Fig EV2G). Importantly, the RNA-IP of Mmi1 R351E and R381E point mutant coupled to PCR (which is more sensitive than the RNA-IP Seq) confirmed that Mmi1 YTH domain specifically recognizes the five new lncRNAs identified by our RNA-IP high-throughput sequencing and computational approaches (Fig 1E and F). We named these lncRNAs non-coding RNA associated to Mmi1 (nam). Hence, from our broad search of Mmi1 RNA targets based on the combination of different approaches, we discovered new RNAs, including euchromatic and heterochromatic lncRNAs. Click here to expand this figure. Figure EV2. Characterization of Mmi1 YTH domain point mutants R351E and R381E Sequence alignment of YTH domains from the yeast Schizosaccharomyces pombe (Sp), the yeast Zygosaccharomyces rouxii (Zr), and Homo sapiens (Hs). Ribbon diagram of Mmi1 YTH domain structure in the same orientation as in Fig 2C. RNA-IP showing that the in vivo binding of Mmi1 to three of its known targets (ssm4, rec8, and spo5 mRNAs) is strongly reduced for R351E and R381E Mmi1 mutants. Gel filtration showing similar elution behavior for WT, R351E, and R381E Mmi1 YTH domains. RNA pull-down showing in vitro that mutations R351E and R381E impair Mmi1 binding to a RNA containing the UUAAAC motif. Relative enrichments of Mmi1 protein recovered after RNA pull-downs done in (E). The quantification was estimated from three independent experiments. See the Materials and Methods for more details. Fluorescence microscopy images showing the cellular localization of Mmi1-R351E, Mmi1-R381E and Mmi1 wild-type (WT) proteins. Nuclear DNA was stained with DAPI (blue). Scale bar, 10 μm. Data information: Average fold enrichment is shown with error bars that indicate mean average deviations (n = 3) for (C, F). Source data are available online for this figure. Download figure Download PowerPoint Mmi1 association with the sole nam1 lncRNA regulates the MAPK-mediated entry into sexual differentiation Mmi1 is well known as an inhibitor of sexual differentiation progression that prevents entry into meiosis by targeting and triggering the degradation of meiotic mRNAs (Harigaya et al, 2006). In response to nutrient starvation (mainly nitrogen), S. pombe cells undergo sexual differentiation (Fig 2A), to allow them to adapt and resist to conditions not favorable to cell growth. At the onset of sexual differentiation, two cells of opposite mating type (h+ and h-) mate to produce a zygote. The zygote then undergoes genome duplication followed by meiosis and the formation of four spores. Quite unexpectedly, we found that mmi1∆ cells poorly execute sexual differentiation, indicating that Mmi1 may be also required to promote sexual differentiation (Figs 2A and EV3A). Further analysis of mmi1∆ cells showed that they poorly mate, indicating that the onset of sexual differentiation is defective (Fig 2B). This defect occurs irrespective of the mating type identity of mmi1∆ cells (Fig EV3B and C). We then assessed the importance of Mmi1 binding to RNA for the control of entry into sexual differentiation by taking advantage of our Mmi1 R351E and R381E point mutants and analyzing whether their expression rescues the cell differentiation defect. While the expression of Mmi1 wild-type protein completely rescues the sexual differentiation of mmi1∆ cells, the expression of Mmi1-R351E or Mmi1-R381E YTH mutant proteins does not (Figs 2A and B, and EV3A), indicating that the binding of Mmi1 YTH domain to one or more RNA is essential for the proper control of entry into sexual differentiation. Figure 2. Mmi1 binding to nam1 lncRNA controls MAPK-mediated entry into sexual differentiation Upper part, scheme of Schizosaccharomyces pombe sexual differentiation. Lower part, microscopy images showing WT and mmi1∆ cells, and mmi1∆ cells expressing Mmi1-R351E or Mmi1-R381E mutant proteins. Images were taken after 24 h of induction of sexual differentiation by growth on SPAS medium. The percentage of cells that underwent differentiation and iodine vapor assays, conducted on the corresponding patches, are shown at the bottom of each image. Scale bar, 10 μm. Mating assay showing the percentage of zygotes forming over time in the same cells as in (A). Scheme of nam1-byr2 locus. Mmi1 UNAAAC binding motifs are depicted by white lines. Scheme of nam1-byr2 locus in nam1-1 cells highlighting the eight UNAAAC motifs mutated (red lines). Mmi1 RNA-IPs showing the specific loss of binding of Mmi1 to nam1-1 lncRNA but not to mei4 mRNA, another target of Mmi1. Mating assay showing the percentage of zygotes formed over time in WT and nam1-1 cells. Western blots showing the level of Flag-Byr2 protein over the first 4 h of sexual differentiation in WT and nam1-1 cells. Tubulin (Tub1) level was used as a loading control. Microscopy images of WT
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