Accurate and extensive regulation of meiotic gene expression is crucial to distinguish germ cells from somatic cells. In the fission yeast Schizosaccharomyces pombe, a YTH family RNA-binding protein, Mmi1, directs the nuclear exosome-mediated elimination of meiotic transcripts during vegetative proliferation. Mmi1 also induces the formation of facultative heterochromatin at a subset of its target genes. Here, we show that Mmi1 prevents the mistimed expression of meiotic proteins by tethering their mRNAs to the nuclear foci. Mmi1 interacts with itself with the assistance of a homolog of Enhancer of Rudimentary, Erh1. Mmi1 self-interaction is required for foci formation, target transcript elimination, their nuclear retention, and protein expression inhibition. We propose that nuclear foci formed by Mmi1 are not only the site of RNA degradation, but also of sequestration of meiotic transcripts from the translation machinery.https://doi.org/10.7554/eLife.32155.001
Regulation of gene expression is fundamental for adaptation to environmental changes in all types of cells. Therefore, gene expression is strictly regulated at all stages from transcription to translation. Regulation of meiotic gene expression is especially crucial to differentiate between the somatic and germ cells. It has been demonstrated that ectopic expression of germline genes causes various cellular defects, including genome instability and tumorigenesis (Folco et al., 2017; Greve et al., 2015; Janic et al., 2010).
Hundreds of genes are upregulated when the fission yeast Schizosaccharomyces pombe cells enter meiosis from the mitotic cell cycle in response to nutrient starvation (Mata et al., 2002). During the mitotic cell cycle, meiotic genes are strictly suppressed by post-transcriptional mechanisms, in addition to transcriptional regulation, since mistimed expression of meiotic genes severely impairs cell growth. A large number of meiosis-specific transcripts carry a specific region called DSR (determinant of selective removal) and are recognized by a YTH family RNA-binding protein, Mmi1, in mitotically growing S. pombe cells. Mmi1 then induces nuclear exosome-mediated RNA elimination (Harigaya et al., 2006; Yamanaka et al., 2010). DSR activity is exhibited by enriched repeats of the hexanucleotide UNAAAC motif (Hiriart et al., 2012; Yamashita et al., 2012). The Mmi1 YTH domain preferentially binds to the unmethylated UNAAAC motif, contrasting with the YTH domains in other organisms including mammals, which selectively bind to N6-methyladenosine-containing RNAs (Chatterjee et al., 2016; Wang et al., 2016; Wu et al., 2017). The DSR region has been found in a group of meiotic transcripts including mei4, which encodes a key meiotic transcription factor (Horie et al., 1998), and ssm4, which encodes a subunit of the dynactin complex (Niccoli et al., 2004). Red1, a zinc-finger protein, is another crucial factor involved in the Mmi1-driven RNA elimination (Sugiyama and Sugioka-Sugiyama, 2011; Yamashita et al., 2013). Red1 constitutes a complex termed MTREC (Mtl1-Red1 core) or NURS (nuclear RNA silencing) with the Mtr4-like RNA helicase, Mtl1, and transfers the Mmi1-bound meiotic transcripts to the nuclear exosome (Egan et al., 2014; Lee et al., 2013; Zhou et al., 2015). In human cells, a similar protein complex, PAXT, composed of a Red1-related zinc-finger protein (ZFC3H1) and an Mtr4 ortholog (hMTR4), has been reported to induce nuclear exosome-dependent RNA degradation (Meola et al., 2016). Recently, ZFC3H1 and hMtr4 have also been shown to prevent nuclear export of non-coding RNAs (Ogami et al., 2017).
Mmi1 forms several dot structures in the nucleus of the mitotically growing cells (Harigaya et al., 2006). Many factors cooperating with Mmi1, including Red1 and exosome subunits, localize to the Mmi1 foci (Sugiyama and Sugioka-Sugiyama, 2011; Yamanaka et al., 2010; Yamashita et al., 2013), suggesting that the foci are the site of degradation of the DSR-containing meiotic transcripts; however, the precise location of the Mmi1 foci in the nucleus remains elusive.
When S. pombe cells initiate meiosis, Mmi1-mediated RNA degradation must be suppressed so that DSR-containing meiotic transcripts are expressed. Downregulation of Mmi1 during meiosis is achieved by sequestration of Mmi1 to a meiosis-specific chromosome-associated dot, Mei2 dot (Harigaya et al., 2006). Mei2 dot is composed of an RNA-binding protein, Mei2, and its binding partner, meiRNA, and is located at the sme2 locus, which encodes meiRNA (Shimada et al., 2003; Watanabe et al., 1997; Yamashita et al., 1998). meiRNA carries the DSR region and is targeted by Mmi1, suggesting that meiRNA serves as a decoy for luring Mmi1 (Shichino et al., 2014; Yamashita et al., 2012).
Mmi1 also plays vital roles in a wide range of regulation of target gene expression, in addition to RNA elimination. Mmi1 induces the assembly of facultative heterochromatin at a subset of its target genes (Hiriart et al., 2012; Tashiro et al., 2013; Zofall et al., 2012). Binding of Mmi1 to target transcripts triggers the recruitment of factors involved in heterochromatin formation along with Red1. It has been demonstrated that a highly conserved multiprotein complex, Ccr4/Not, participates in the Mmi1-mediated heterochromatin formation, albeit to a limited extent (Cotobal et al., 2015; Sugiyama et al., 2016). Mmi1 has also been shown to accelerate deadenylation by Ccr4/Not, although its in vivo significance remains unknown (Stowell et al., 2016). Furthermore, Mmi1 regulates the termination of transcription of its target genes (Chalamcharla et al., 2015; Shah et al., 2014; Touat-Todeschini et al., 2017). Our recent study has revealed that Mmi1-mediated termination of an upstream non-coding RNA ensures the expression of downstream genes (Touat-Todeschini et al., 2017).
Mmi1-mediated multilevel regulation is obviously important for proper gene expression in the mitotically growing cells. However, the mechanism of Mmi1-mediated regulation is still not completely known. In this study, we report that Mmi1 prevents the mistimed expression of meiotic proteins by tethering their mRNAs to the nuclear foci. Furthermore, we demonstrated that Mmi1 interacts with itself through the N-terminal region, assisted by a highly conserved cofactor, Erh1. Mmi1 self-interaction is critical for the nuclear retention of meiotic transcripts and consequent prevention of their translation. These results revealed a novel facet of Mmi1-mediated regulation.
Disruption of mmi1 results in a severe growth defect due to the aberrant expression of meiotic transcripts. This defect was alleviated by the deletion of mei4 (Harigaya et al., 2006), indicating that mei4 is a crucial target of Mmi1-mediated elimination. red1∆ cells inappropriately accumulate meiotic transcripts such as mei4 and ssm4 during vegetative growth, but are viable albeit showing cold and weak high-temperature sensitivity (Figure 1A,B, Figure 1—figure supplement 1A, and Figure 1—figure supplement 2A,B) (Sugiyama and Sugioka-Sugiyama, 2011; Yamashita et al., 2013). Deletion of mei4 did not suppress this temperature-sensitivity (Figure 1A), suggesting that these defects are independent of meiotic transcript accumulation. This was also the case in a temperature-sensitive mutant of rrp6, which encodes a catalytic subunit of the nuclear exosome (Figure 1A). To analyze the discrepancy between growth profiles of mmi1 and red1 mutants, we investigated the expression of transcripts and proteins of mei4 in temperature-sensitive mmi1-ts3 and red1∆ cells. mei4 transcripts were ectopically accumulated in both mutants during the mitotic cell cycle (Figure 1B and Figure 1—figure supplement 2A). Strikingly, Mei4 protein expression was hardly detected in red1∆ cells, but accumulated in cells with reduced Mmi1 activity (Figure 1C). In the temperature-sensitive rrp6-32 mutant, expression of Mei4 protein was suppressed, whereas mei4 transcripts accumulated, as seen in red1∆ cells (Figure 1B,C and Figure 1—figure supplement 2A). Mei4 protein was detected in both mmi1-ts3 red1∆ and mmi1-ts3 rrp6-32 double mutants, indicating that Mei4 protein expression requires both transcript accumulation and Mmi1 inactivation. Similarly, protein expression of ssm4, another DSR-containing gene, was detected when Mmi1 activity was dampened (Figure 1—figure supplement 1B). These results suggest that Mmi1 likely inhibits the expression of proteins encoded by ectopically accumulated DSR-containing mRNAs.
Mmi1 is shown to regulate the transcription termination of its targets (Chalamcharla et al., 2015; Shah et al., 2014; Touat-Todeschini et al., 2017). Northern blot analysis revealed that mei4 and ssm4 transcripts in mmi1-ts3 cells were similar in size to the wild-type meiotic counterparts (Figure 1—figure supplement 2C,D), suggesting that Mmi1 has little, if any, impact on the termination of these genes. In red1∆ or rrp6-32 cells, a slight increase in the size of both mei4 and ssm4 transcripts was observed (Figure 1—figure supplement 2A,B), thereby suggesting a role of these factors in the regulation of termination, including polyadenylation. In cells with impaired Red1 or Rrp6 function, longer ssm4 transcripts accumulated (Figure 1—figure supplement 2B), which were seen to comprise of ssm4 and its upstream gene (Zofall et al., 2012).
Since Mmi1 specifically interacts with DSR-containing transcripts and localizes to nuclear foci (Harigaya et al., 2006; Yamashita et al., 2012), we hypothesized that Mmi1 may capture DSR-containing transcripts in the nuclear foci and sequester them from the translation machinery, even when selective RNA degradation is impaired. We examined the cellular distribution of mei4 and ssm4 transcripts using single-molecule RNA fluorescence in situ hybridization (smFISH). We detected a concentration of mei4 and ssm4 transcripts in the nucleus in red1∆ or rrp6-32 cells (Figure 2A,B and Figure 2—figure supplement 1A,B). In red1∆ and rrp6-32 cells, most nuclear RNA spots were larger than cytoplasmic spots, suggesting that multiple molecules of mei4 or ssm4 transcripts could have converged in the nucleus. Because we counted each such large spot as one, the number of nuclear spots may possibly be underestimated in red1∆ and rrp6-32 cells. In contrast, when the Mmi1 function was compromised, these transcripts were exported from the nucleus. These observations support the idea that Mmi1 impedes the translation of DSR-containing meiotic transcripts by retaining them in the nucleus. The cytoplasmic distribution of mei4 and ssm4 transcripts in mmi1 mutant cells was halted by the inactivation of Rae1 (Figure 2C,D and Figure 2—figure supplement 1C,D), which is an essential factor in the nuclear export of mRNAs (Brown et al., 1995; Yoon et al., 1997). This suggests that the nuclear export of DSR transcripts is mediated by the canonical mRNA export pathway. Elevation of mei4 and ssm4 transcript levels was observed in rae1-167 cells, as shown previously (Sugiyama et al., 2013).
To gain further insight into the mechanism underlying the nuclear retention of DSR-containing transcripts, we visualized the localization of reporter transcripts (comprised of U1A tag, the firefly luciferase open reading frame, and 0–24 copies of the DSR motif) by coexpressing U1A-YFP (Figure 3A). We previously demonstrated that more than 5 copies of the DSR motif exert DSR activity (Yamashita et al., 2012). Transcripts carrying eight and more DSR motifs in our reporter system were degraded and decay fragments containing U1A tag were observed, whereas transcripts carrying four motifs were stably expressed (Figure 3—figure supplement 1A). We detected foci in mitotic cells expressing the reporter constructs with eight and more DSR motifs (Figure 3B,C and Figure 3—figure supplement 1B). These findings indicate that the observed foci may have formed from the degraded fragments of reporter transcripts carrying U1A tag. Foci formation took place inside the nucleus, confirmed by simultaneous observation of the nucleoporin Nup60 (Figure 3D). Foci were scarcely observed in cells expressing transcripts containing 0 and 4 DSR motifs, suggesting that, unlike smFISH, transcripts could be detected in this system only when a certain concentration of molecules were gathered. The frequency of cells exhibiting foci and the number of foci per cell increased with the number of DSR motifs (Figure 3C). DSR-transcript foci coincided with Mmi1 (Figure 3E). Foci formation was severely impaired in mmi1∆ cells, although the transcripts escaped degradation and accumulated (Figure 3F,G and Figure 3—figure supplement 2A,B,C). These observations suggest that DSR transcripts were dispersed in the absence of Mmi1. In red1∆ and rrp6-32 cells, in which degradation of DSR transcripts was compromised, foci became prominent, and the frequencies of cells carrying four and more foci increased (Figure 3F,G and Figure 3—figure supplement 2A,B,C). These foci also colocalized with Mmi1 inside the nucleus (Figure 3—figure supplement 2D,E).
Next, we investigated the mechanism by which Mmi1 forms nuclear foci. Mmi1 lacking the C-terminal YTH domain (Mmi1-∆YTH) showed similar localization to that of full-length Mmi1 (Figure 4A,B). We then attempted to determine the region responsible for foci formation in the N-terminus of Mmi1, and found that the domain of residues 61–180 had the ability to form nuclear foci (Figure 4B and Figure 4—figure supplement 1A). Two-hybrid analyses and immunoprecipitation assays indicated that Mmi1 interacted with itself through this domain (Figure 4C,D and Figure 4—figure supplement 1B,C), so we designated this region SID (self-interaction domain). Mmi1 lacking SID (Mmi1-∆SID) retained the ability to bind to the DSR region (Figure 4—figure supplement 1D). However, Mmi1-∆SID lost the function to induce the elimination of DSR-containing transcripts (Figure 4E and Figure 4—figure supplement 1E) and could not rescue the growth defect of temperature-sensitive mmi1 mutant cells (Figure 4F). It was also noted that expression of Mmi1-∆YTH had a dominant-negative effect (Figure 4E,F and Figure 4—figure supplement 1E). Deletion of SID also resulted in loss of interaction with Red1 (Figure 4G), suggesting that Mmi1 interacts with factors involved in Mmi1-mediated elimination through SID.
Next, we assessed whether removing SID from Mmi1 affects the nuclear retention and expression of DSR-containing transcripts. mei4 and ssm4 transcripts were mainly observed in the cytoplasm of mmi1 mutant cells expressing Mmi1-∆SID, as cells expressing Mmi1-∆YTH (Figure 5A,B and Figure 5—figure supplement 1A,B). Nuclear foci of the reporter transcripts were dispersed in cells expressing Mmi1-∆SID (Figure 5—figure supplement 2A,B), although full-length transcripts accumulated (Figure 5—figure supplement 2C). Correspondingly, both mRNAs and proteins of mei4 and ssm4 were expressed strongly in mmi1-ts3 cells expressing Mmi1-∆SID (Figure 4E, Figure 4—figure supplement 1E, Figure 5C and Figure 5—figure supplement 3). In mmi1-ts3 cells expressing Mmi1-∆YTH, Mei4 protein was expressed at a permissive temperature, confirming the dominant-negative effect of Mmi1-∆YTH (Figure 5C). These observations indicate that SID is crucial for tethering DSR-containing meiotic transcripts to nuclear foci.
To identify factors involved in the foci formation of Mmi1, we next examined the localization of Mmi1 in deletion mutants lacking genes encoding factors related to Mmi1-mediated elimination, including Red1 (Sugiyama and Sugioka-Sugiyama, 2011; Sugiyama et al., 2012; Yamashita et al., 2013). The deletion of red1, rhn1/iss4, and iss9 had no impact on Mmi1 foci formation (Figure 6—figure supplement 1A). Rhn1 and Iss9 also did not colocalize with Mmi1 (Figure 6—figure supplement 1B). In cells lacking erh1, which encodes a homolog of Enhancer of Rudimentary (Krzyzanowski et al., 2012; Yamashita et al., 2013), the frequency of cells carrying Mmi1 foci was decreased (Figure 6A). Erh1 colocalized and interacted with Mmi1 (Figure 6B,C), as shown previously (Sugiyama et al., 2016). The interaction between Erh1 and Mmi1 was impaired when Mmi1 lacked SID (Figure 6C). Furthermore, Mmi1 self-interaction was severely dampened, although not completely abrogated, in erh1∆ cells (Figure 6D) while the deletion of erh1 had no effect on the interaction between Mmi1 and Red1 (Figure 6—figure supplement 1C). These evidences suggest that Erh1 might be engaged in Mmi1-mediated elimination, by binding to SID and reinforcing the self-interaction and foci formation of Mmi1, although the self-interaction may be induced by Mmi1 itself. This is consistent with the two-hybrid analysis described above (Figure 4C and Figure 4—figure supplement 1B,C).
Next, we examined the distribution of DSR-containing transcripts in erh1∆ cells. mei4 and ssm4 transcripts were exported to the cytoplasm, as cells lacking SID (Figure 5A,B and Figure 5—figure supplement 1A,B). In erh1∆ cells, nuclear foci formation of the reporter transcripts was also severely impaired (Figure 5—figure supplement 2A,B). Transcripts and proteins of mei4 and ssm4 were accumulated in erh1∆ cells, although to a lesser extent than in mmi1-ts3 cells (Figure 6E,F and Figure 6—figure supplement 1D,E). The erh1∆ cells displayed cold sensitivity (Sugiyama et al., 2016; Yamashita et al., 2013), and this growth defect was suppressed by the deletion of mei4 (Figure 6G), as was the case in mmi1 deletion. From these observations, we conclude that DSR-containing transcripts are retained at nuclear foci through Mmi1 self-interaction, which is assisted by Erh1.
Recently, we have shown that Mei4 protein is expressed in cells lacking Pab2, a nuclear poly(A)-binding protein, which acts in Mmi1-mediated RNA degradation (Cotobal et al., 2015). We compared the expression levels of Mei4 protein in mmi1-ts3, red1∆, and pab2∆ cells (Figure 6—figure supplement 2). In pab2∆ cells, Mei4 was expressed very weakly, at a level similar to that in red1∆ cells. This indicates that Pab2 does not play a significant role in Mmi1-driven nuclear tethering of DSR-containing transcripts.
Our data demonstrated that Mmi1 controls the expression of meiotic genes in a multilayered fashion, namely through heterochromatin silencing, selective elimination of transcripts, and prevention of protein expression by nuclear foci tethering (Figure 7). The meiotic transcripts carrying DSR are recognized by Mmi1, localized in nuclear foci through Mmi1 self-interaction, and degraded by the nuclear exosome. Induction of RNA degradation might be a primary function of Mmi1. However, even though RNA degradation is impaired, nuclear export of the DSR-containing transcripts is prevented by Mmi1 and consequently their protein expression is suppressed. Transcription of some Mmi1-target gene loci, including mei4, is also silenced through Mmi1-mediated facultative heterochromatin formation (Hiriart et al., 2012; Tashiro et al., 2013; Zofall et al., 2012). In the red1 and rrp6 mutant cells, degradation of meiotic transcripts is dampened. However, transcripts are tethered to the nuclear Mmi1 foci and their expression is suppressed through sequestration from the translation machinery. In the absence of Mmi1, degradation of the DSR-containing meiotic transcripts is compromised and they are exported to the cytoplasm and translated, leading to the deleterious expression of meiotic genes. Recently, ZFC3H1, a possible counterpart of Red1 in human cells, has been demonstrated to induce nuclear exosome-mediated degradation of long noncoding RNAs, and prevent their nuclear export in cooperation with hMtr4, an ortholog of which forms a complex with Red1 (Ogami et al., 2017). hMtr4 has also been shown to induce degradation of mRNAs by inhibiting their nuclear export and recruiting the nuclear exosome (Fan et al., 2017). It is an intriguing question whether a target-recognition factor, equivalent to Mmi1, works in human cells, although Red1 and ZFC3H1 act differently in nuclear export of the target transcripts.
Regulation of protein expression plays a critical role during meiosis in other organisms. During oogenesis in Xenopus laevis, a small subset of mRNAs, such as mRNAs encoding key regulators for meiotic progression, are translationally activated, whereas other mRNAs are repressed (Sheets et al., 2017). Changes in poly(A) tail length are known to be important for this regulation (Weill et al., 2012). In addition, it has been suggested that the N6-methyladenosine modification, which is often recognized by the YTH domain, is involved in this translational regulation (Qi et al., 2016). In S. pombe, ribosome profiling has also shown that translation efficiency of the DSR-containing transcripts is indeed upregulated during meiosis (Duncan and Mata, 2014). This is consistent with our findings that Mmi1 modulates gene expression profiles, between mitosis and meiosis, by regulating protein expression through nuclear tethering of meiotic mRNAs, in addition to controlling their stability.
The DSR-containing meiotic transcripts localize in the nuclear Mmi1 foci, where many other factors involved in selective elimination exist (Sugiyama and Sugioka-Sugiyama, 2011; Yamanaka et al., 2010; Yamashita et al., 2013), implying that Mmi1 foci are the major sites of meiotic transcript degradation. The Mmi1 foci are distinct from the mei4 locus, which encodes a key target of Mmi1 (Egan et al., 2014; Shichino et al., 2014), while enrichment of Mmi1 at the mei4 locus has been observed by chromatin immunoprecipitation analyses (Chalamcharla et al., 2015; Tashiro et al., 2013). It is possible that Mmi1 co-transcriptionally recognizes its target transcripts at their gene loci and Mmi1-transcript complexes move to the nuclear foci where RNA degradation takes place; however, we cannot exclude the possibility of Mmi1-inducing RNA degradation on its target gene loci. Further investigation would be required to determine the site of RNA degradation and the precise localization of the Mmi1 foci.
Mmi1 interacts with itself through the N-terminal SID region with assistance from Erh1. Mmi1 self-interaction is likely to trigger nuclear foci formation, which may be a prerequisite for target transcript degradation and their nuclear tethering. The following observations support this proposal: SID is required for foci formation; Mmi1 lacking SID loses its function of inducing degradation of the target transcripts and preventing their protein expression; erh1∆ cells, in which Mmi1 self-interaction is impaired, have less Mmi1 foci and show weakened Mmi1 activity, although the interaction between Mmi1 and Red1 is intact. Mmi1 forms foci exclusively in the nucleus. This may be because Mmi1 foci are formed only when a certain number of Mmi1 molecules are enriched in a limited area. However, the possibility of a nuclear-specific factor(s), essential for Mmi1 foci formation, cannot be completely excluded. Further studies would be needed to fully elucidate the mechanism underlying self-interaction and nuclear foci formation of Mmi1.
Erh1 is highly conserved among eukaryotes. Orthologs of Erh1 have been shown to play roles in various processes, including transcriptional and post-transcriptional regulation (Weng and Luo, 2013). It is an intriguing question whether ERH proteins play a similar role as the S. pombe Erh1.
Foci formation by the YTH family proteins is known in cells of higher eukaryotes. YTHDC1/YT521-B, which controls mRNA splicing (Lence et al., 2016; Xiao et al., 2016), interacts with itself and forms nuclear foci called YT bodies (Hartmann et al., 1999; Nayler et al., 2000). The cytoplasmic YTH protein YTHDF2, which regulates the stability of transcripts (Wang et al., 2014), also aggregates into the cytoplasmic foci and interacts with the processing body, which is enriched in the factors involved in RNA degradation (Wang et al., 2014). These YTH-containing proteins have regions that are enriched in particular amino acids (proline, arginine, and glutamic acid in YTHDC1; proline, glutamine, and asparagine in YTHDF2) and these regions are crucial for proper localization of proteins (Hartmann et al., 1999; Wang et al., 2014). The SID region of Mmi1 is also enriched in serine (20/120 amino acids), arginine (12/120), tyrosine (12/120), and proline (15/120). Most YTH-family proteins do not share sequence similarity without the YTH domain. It is possible that some of them share structural features, such as low complexity regions, and have the ability to form foci through those regions.
Rigorous Mmi1-mediated regulation may function to remove meiotic factors harmful for mitotic growth. Growth retardation of mmi1 mutant cells is mainly caused by inappropriate accumulation of the Mei4 protein during the mitotic cell cycle. It has also been reported that the ectopic expression of meiotic cohesin rec8, induced by the deletion of mmi1, causes abnormality in mitotic chromosome segregation (Folco et al., 2017). In higher eukaryotes, certain genes are exclusively expressed in germ lines and cancerous tissues. Such genes, named cancer/testis antigen genes, include the meiotic cohesin genes, counterparts of which are targeted by Mmi1 in S. pombe (Feichtinger et al., 2012; Simpson et al., 2005). It would be interesting and important to investigate whether similar mechanisms of Mmi1-mediated regulation operate in other organisms.
The S. pombe strains used in this study are listed in Supplementary file 1. Complete medium YE, minimal medium SD and MM (Moreno et al., 1991), synthetic sporulation medium SSA (Egel and Egel-Mitani, 1974), and sporulation agar SPA (Gutz et al., 1974) were used. The general genetic methods used to analyze the S. pombe strains have been previously described (Gutz et al., 1974). Standard gene-targeting protocols were carried out to create deletion mutants and epitope-tagged strains (Bähler et al., 1998; Sato et al., 2005). Truncated alleles of mmi1 were constructed using the PrimeSTAR Mutagenesis Basal Kit (Takara, Japan). The co2 region, a gene-free region on chromosome I, was selected as a target site for chromosomal integration of cloned constructs (Kakui et al., 2015).
S. pombe cells were lysed by vortexing vigorously with glass beads in a buffer (0.2 M Tris-HCl [pH7.5], 0.5 M NaCl, 0.01 M ethylenediaminetetraacetic acid (EDTA), 1% sodium dodecyl sulfate (SDS)). After two rounds of extraction with phenol:chloroform:isoamyl alcohol (25:24:1), RNA was recovered by ethanol precipitation. Ten µg of total RNA was denatured with formamide, separated by electrophoresis on a 1.2% agarose gel containing 1% formaldehyde, and blotted onto a GeneScreen Plus membrane (Perkin Elmer, Waltham, Massachusetts). DNA fragments to probe transcripts were amplified by PCR using primers (Supplementary file 2) and labeled with [γ-32P]dCTP using MegaPrime DNA Labeling System (GE Healthcare, UK). In Figure 1—figure supplement 2, RNA was blotted onto a Hybond N + membrane (GE healthcare). DNA fragments to probe the transcripts were amplified by PCR, using suitable primers (Supplementary file 2), and DIG-labeled RNA probes were synthesized using the DIG RNA labeling kit (Roche, Switzerland).
Total RNA was treated with DNase (Turbo DNA-free, Ambion, Waltham, Massachusetts) and reverse-transcribed to cDNA by using ReverTra Ace qPCR Master Mix (TOYOBO, Japan). Quantitative PCR was performed using LightCycler96 (Roche) with SYBR Premix Ex Taq II (Tli RNaseH Plus) (Takara). Primers used in this study are listed in Supplementary file 2.
Harvested cells were lysed by vortexing vigorously with glass beads in HB buffer (25 mM MOPS (pH7.2), 5 mM ethylene glycol bis-(2-aminoehylether) tetraacetic acid (EGTA) (pH7.2), 15 mM MgCl2, 150 mM KCl, 50 mM beta-glycerophosphate, 15 mM p-nitrophenylphosphate, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.1 mM Na3VO4, 0.2% NP-40, protease inhibitor cocktail (Complete Mini EDTA-free, Roche)). The following antibodies were used to detect target proteins: rabbit anti-HA Y11 (Santa Cruz, Dallas, Texas), mouse anti-GFP (Roche), rabbit anti-GFP (Life Technologies, Carlsbad, California), mouse anti-γ-tubulin (Sigma), rabbit peroxidase anti-peroxidase soluble complex antibody (Sigma, St. Louis, Missouri), and mouse anti-Myc 9E10 (Santa Cruz).
Native cell extracts were incubated with antibody-conjugated magnetic beads for 1 hr at 4°C. The beads were then washed three times. Mouse IgG beads (Cell Signaling Technology, Danvers, Massachusetts) and mouse anti-HA beads (MBL, Japan) were used to precipitate TAP-tagged Mmi1 and HA-tagged Mmi1 variants, respectively. Mouse anti-DDDDK beads (MBL) were used as controls.
To observe mitotically growing cells, cells were grown in the logarithmic phase in YE or SD liquid medium at the appropriate temperatures. Imaging was performed with the DeltaVision-SoftWoRx system (GE Healthcare). For live-cell imaging, cells were mounted onto glass bottom culture dishes (MatTek, Ashland, Massachusetts) precoated with lectin, and the dishes were filled with liquid medium. For fixed-cell imaging, cells were mounted on coverslips. Images were acquired as serial sections along the z-axis at intervals of 0.5 µm (for living cell) or 0.3 µm (for fixed cell). All images were deconvolved and stacked using the quick projection algorithm in the SoftWoRx software.
Single-molecule RNA fluorescence in situ hybridization (smFISH) was performed as previously described (Castelnuovo et al., 2013; Heinrich et al., 2013) using mixtures of DNA probes (Supplementary file 3) coupled to Quaser 570 fluorophore (Stellaris, Biosearch Technologies, Petaluma, California). Probes were targeted against the ORF of the mei4 gene or the ssm4 gene.
The image processing procedure comprises the following three steps: (1) mRNA spot extraction, (2) nuclei region segmentation, and (3) cell region segmentation.
In step (1), first, local maxima (intensity peaks) in the fluorescence micrographs of mRNAs were detected as the candidates of mRNA spots and the positions of the local maxima were determined. The local maxima were represented as 1-pixel regions via the above process. Second, these local maxima, which have a higher intensity value than the threshold value, were selected as mRNA spots. The threshold value was determined by an empirical analysis based on the distribution of fluorescence intensities in mei4∆ micrographs, which were selected as reference (control) samples. For micrograph series of ssm4 mRNAs, optimum threshold value was determined empirically by visual inspection of each set of micrographs.
In step (2), first, the fluorescence micrograph of nuclei was blurred using a Gaussian filter, and the blurred image was segmented by an automatic local thresholding technique (Phansalkar et al., 2011). Second, small isolated regions, which were regarded as noise, were removed by an area opening operation (Vincent, 1993).
In step (3), cell region segmentation was performed by a semi-automatic approach. First, the bright field micrograph of cells was blurred using a Gaussian filter to remove noise and smoothen the cell region. Second, edge detection was performed, and the resultant image was binarized. Further, small isolated regions were removed by the area opening operation. Third, a closing operation (Soille, 2003) was applied to the binarized image to fill holes and cracks in the binarized cell region. When adjacent or aggregated cell regions were clustered, it was difficult to separate them automatically. Therefore, the contact areas of cells were manually separated, and the shape of the segmented cell region was restored by a region growing technique (Soille, 2003) and manual editing.
Finally, we measured the ratio of the number of spots in the nuclei region to that in the cytoplasmic region. This cytoplasmic region is obtained by subtracting the nuclei region from the cell region.
To visualize DSR-containing transcripts in living cells, the human snRNP protein U1A was used, as it specifically recognizes the stem-loop structure of U1 snRNA (Andoh et al., 2006; Takizawa and Vale, 2000). The constitutive adh1 promoter, four copies of an U1 snRNA stem-loop sequence, the open-reading frame (ORF) of the firefly luciferase gene (luc), and the terminator of the nmt1 gene were cloned, and tandem repeats of the hexanucleotide DSR motif (TTAAAC) were inserted between the luc gene and the nmt1 terminator. The cloned construct was inserted at the co2 region on chromosome I. The YFP-tagged U1A protein was expressed from the arg1 gene locus under the control of the adh41 promoter, which carried mutations in the TATA sequence of the adh1 promoter (TATAAATA to ATAAA).
The mmi1 ORF or its truncated versions were cloned in pGBKT7 or pGADT7 (Clontech, Mountain View, California). The Saccharomyces cerevisiae strain AH109 (MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4Δ, gal80Δ, LYS2::GAL1UAS-GAL1TATA-HIS3, MEL1, GAL2UAS-GAL2TATA-ADE2, URA3::MEL1UAS-MEL1TATA-lacZ) was transformed with the plasmids. pGADT7-T-antigen and pGBKT7-p53 were used as controls.
An RNA electrophoretic mobility shift assay was performed as previously described (Yamashita et al., 2012) with modifications. Bacterially purified proteins were preincubated at room temperature with heparin sulfate (5 mg/mL at final concentration) for 20 min to prevent non-specific binding. The proteins were mixed with the RNA probe labeled by digoxigenin (DIG) and incubated at room temperature (23–27°C) for 30 min. Samples were electrophoresed on 6% acrylamide:bisacrylamide (29:1) gel for 3 hr at 150 V and electroblotted to GeneScreen Plus membrane (Perkin Elmer) using 0.5 × tris–borate–EDTA buffer.
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Torben Heick JensenReviewing Editor; Aarhus University, Denmark
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
Thank you for submitting your article "YTH-RNA-binding protein tethers meiotic transcripts to prevent deleterious translation" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by James Manley as the Senior Editor. The reviewers have opted to remain anonymous.
The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.
The manuscript by Yamashita et al. suggests that Mmi1 tethers meiotic transcripts to nuclear foci and thereby inhibits nuclear export and translation of this class of mRNAs. In addition, the authors illustrate that with the assistance of Erh1, Mmi1 interacts with itself and that this self-interaction is required for nuclear foci formation, target degradation and translation suppression. Overall, the manuscript is well written and the experiments are well conducted. Still improvements are needed as outlined below:
1) A major concern is the way in which the authors talk about the role of Mmi1 in 'translational suppression'. In several parts of the manuscript, the impression is given that this is a direct role of the protein (see for example the title, parts of the Abstract, and in the Discussion). This is misleading. In fact, the authors show that Mmi1 has a role in preventing mRNA export, indicating that any effects on protein levels are likely to be indirect. Indeed, the conclusion on nuclear export in the mmi1 mutant might even be an indirect consequence of defects in Mmi1-dependent nuclear retention and nuclear decay. The question is whether the primary function of Mmi1 is to retain meiotic transcript in the nucleus to prevent cytoplasmic translation and/or to promote nuclear decay at the site of transcription. These issues need to be clarified by more careful interpretation and discussion of the data.
2) Figure 1: The authors use RT-qPCR to measure the levels of mei4-TAP and ssm4-TAP mRNAs. Yet, Mmi1 was recently shown to influence transcription termination. It would therefore be important to confirm that mei4 and ssm4 mRNAs accumulating in mmi1 and red1 mutants have similar 3' ends and poly(A) tail length, both of which are known to influence nuclear export and cytoplasmic translation. Accordingly, the authors should analyze mei4 and ssm4 transcripts by Northern blotting and/or RNase cleavage assays to examine the 3' end of these mRNAs.
3) The last part of the manuscript, which describes Mmi1 function in the pairing of homologous chromosomes, is somewhat out of context with the rest of the paper. To tie it better together, the authors could provide more data e.g. by examining the mmi1 domain deletion strains and/or explain/discuss more explicitly why these results are important in the scope of this manuscript. Alternatively, the data could simply be left out of the manuscript.https://doi.org/10.7554/eLife.32155.032
- Akira Yamashita
- Akira Yamashita
- Akira Yamashita
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We thank S Hauf and D Zenklusen for sharing the FISH protocol; T Tani for providing the RNA visualizing system; D Duncan and J Mata for providing the mei4-TAP strain; Center for Radioisotope Facilities, Okazaki Research Facilities, NINS and A Nakade for technical support. This work was supported by JSPS KAKENHI Grant Number 15H04333 to AY, a Grant from The Naito Foundation to AY and a Grant for Basic Science Research Projects from The Sumitomo Foundation (Grand Number 140283) to AY.
- Torben Heick Jensen, Reviewing Editor, Aarhus University, Denmark
- Received: September 20, 2017
- Accepted: January 31, 2018
- Version of Record published: February 9, 2018 (version 1)
© 2018, Shichino et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.