Defining the Mer1 and Nam8 meiotic splicing regulons by cDNA rescue

Abstract

Meiosis-specific pre-mRNA splicing in budding yeast embraces multiple pre-mRNA targets grouped into regulons defined by their genetic requirements for vegetatively optional splicing factors (e.g., splicing enhancer Mer1 and the U1 snRNP subunit Nam8) or snRNA modifications (trimethylguanosine caps). Here, we genetically demarcate a complete meiotic splicing regulon by the criterion of cDNA bypass of the requirement for the governing splicing regulators to execute sporulation. We thereby show that the Mer1 and Nam8 regulons embrace four essential pre-mRNAs: MER2, MER3, SPO22, and AMA1. Whereas Nam8 also regulates PCH2 splicing, PCH2 cDNA is not needed for sporulation by nam8D diploids. Our results show that there are no essential intron-containing RNAs missing from the known roster of Mer1 and Nam8 targets. Nam8 is composed of three RRM domains, flanked by N-terminal leader and C-terminal tail segments. We find that the RRM2 and RRM3 domains, and their putative RNA-binding sites, are essential for yeast sporulation, whereas the leader, tail, and RRM1 modules are not.

Genetic interactions of hypomorphic mutations in the m7G cap-binding pocket of yeast nuclear cap binding complex: An essential role for Cbc2 in meiosis via splicing of MER3 pre-mRNA

“…As shown previously (Qiu et al 2011c), the working definition of a ''complete'' meiotic splicing regulon is the ability to rescue the sporulation defect caused by mutation of the splicing ''governor'' by expressing intronless cDNA versions of the essential meiotic RNAs that are targeted by the governor (which, in this case, are SAE3 and MER3). Therefore, we introduced intronless cSAE3 and cMER3 (under the control of their native promoters) into the chromosomal leu2ThisG locus of cbc2-ND42 SKY diploids and then assessed their sporulation efficiency.…”

“…Introduction of SAE3 and MER3 cDNAs at the leu2ThisG locus The cSAE3 and cMER3 genes, consisting of the intronless SAE3 and MER3 cDNAs plus flanking segments of genomic DNA, were described previously (Qiu et al 2011c). The cSAE3 and cMER3 genes were restricted at terminal sites and inserted individually or in tandem (with cSAE3 upstream of cMER3) into yeast integrative vector pRS305 (LEU2) to yield pRS305-cSAE3, pRS305-cMER3, and pRS305-cSAE3-cMER3.…”

Regulated pre-mRNA splicing: The ghostwriter of the eukaryotic genome

“…Under conditions of nutrient starvation diploid yeast cells enter meiosis and sporulate. As with ribosome biosynthesis, this process is tightly regulated at the transcriptional level [63] as well as at the level of splicing [64, 65]. Consistent with this, a number of intron-containing meiotic transcripts can be detected in vegetative cells, but these remain unspliced [42].…”

A dominant role for meiosis-specific 3′ RNA processing in controlling expression of a fission yeast cyclin gene

“…However, the underlying regulatory mechanisms appear to be distinct. In S. cerevisiae, the MER2, MER3, SPO22, and AMA1 transcripts form a regulon controlled positively by Mer1p, a KH domain RNA binding protein expressed only during meiosis (Spingola and Ares 2000;Munding et al 2010;Qiu et al 2011a). These four pre-mRNAs, together with PCH2, require the U1 snRNP protein Nam8p for splicing (Qiu et al 2011a), and PCH2 shares with SAE3 a requirement for trimethylation of the guanosine caps on spliceosomal snRNAs (Qiu et al 2011b).…”

“…In S. cerevisiae, the MER2, MER3, SPO22, and AMA1 transcripts form a regulon controlled positively by Mer1p, a KH domain RNA binding protein expressed only during meiosis (Spingola and Ares 2000;Munding et al 2010;Qiu et al 2011a). These four pre-mRNAs, together with PCH2, require the U1 snRNP protein Nam8p for splicing (Qiu et al 2011a), and PCH2 shares with SAE3 a requirement for trimethylation of the guanosine caps on spliceosomal snRNAs (Qiu et al 2011b). In contrast to the intronic splicing enhancers that act on nearby 59 splice sites in budding yeast, the three fission yeast transcripts in which the signals that govern meiosis-specific splicing have been mapped (mes1, crs1, and rem1) are regulated by sequences outside the coding regions, far from the introns they regulate (Shimoseki and 1 Shimoda 2001;Averbeck et al 2005;Moldon et al 2008).…”