N6-methyladenosine (m6A) reader Pho92 is recruited co-transcriptionally and couples translation to mRNA decay to promote meiotic fitness in yeast

Abstract

N6-methyladenosine (m6A) RNA modification impacts mRNA fate primarily via reader proteins, which dictate processes in development, stress, and disease. Yet little is known about m6A function in Saccharomyces cerevisiae, which occurs solely during early meiosis. Here we perform a multifaceted analysis of the m6A reader protein Pho92/Mrb1. Cross-linking immunoprecipitation analysis reveals that Pho92 associates with the 3’end of meiotic mRNAs in both an m6A-dependent and independent manner. Within cells, Pho92 transitions from the nucleus to the cytoplasm, and associates with translating ribosomes. In the nucleus Pho92 associates with target loci through its interaction with transcriptional elongator Paf1C. Functionally, we show that Pho92 promotes and links protein synthesis to mRNA decay. As such, the Pho92-mediated m6A-mRNA decay is contingent on active translation and the CCR4-NOT complex. We propose that the m6A reader Pho92 is loaded co-transcriptionally to facilitate protein synthesis and subsequent decay of m6A modified transcripts, and thereby promotes meiosis.

m6A-ELISA, a simple method for quantifyingN6-methyladenosine from mRNA populations

“…The standard curve corrected for the background signal, and we observed more than 30-fold signal to noise ratio (Figure 2B). The signal-to-noise ratios obtained with standard curve corrected sample quantifications were comparable to m6A measurements obtained using LC-MS (Figure 2C) (Varier et al 2022).…”

“…Importantly, diploid cells harbouring an IME4 gene deletion display no detectable levels of m6A thus forming the ideal negative control for optimizing the m6A-ELISA protocol (Schwartz et al 2013a). In yeast meiosis, m6A levels are at most 0.1% (Agarwala et al 2012;Varier et al 2022). First, we assessed whether m6A levels could be detected using the ABClonal-A19841 antibody (Figure 1A, 1B, and Supplementary Figure 1A).…”

“…Entry into meiosis was induced in the yeast SK1 diploid cells as previously described, following a standard protocol (Moretto et al 2021). The wild-type and ime4 strains (FW1511 and FW7030) were previously described (Varier et al 2022).…”

The yeast RNA methylation complex consists of conserved yet reconfigured components with m6A-dependent and independent roles

“…Cells were grown in YPD (1.0% (w/v) yeast extract, 2.0% (w/v) peptone, 2.0% (w/v) glucose, and supplemented with uracil (2.5 mg/l) and adenine (1.25 mg/l)). To induce meiosis a standard protocol for sporulation was followed as described previously (Varier et al, 2022) . In short, cells were grown until saturation for 24h in YPD, then grown in pre-sporulation medium BYTA (1.0% (w/v) yeast extract, 2.0% (w/v) bacto tryptone, 1.0% (w/v) potassium acetate, 50 mM potassium phthalate) for about 16 h, and shifted to sporulation medium (SPO) (0.3% (w/v) potassium acetate and 0.02% (w/v) raffinose)).…”

“…More than 1000 mRNAs are methylated during early yeast meiosis (Schwartz et al, 2013). Like in mammals, m6A modified mRNAs are turned over more rapidly compared to unmodified mRNAs (Bushkin et al, 2019;Scutenaire et al, 2022;Varier et al, 2022). The decay of m6A transcripts requires active translation and is mediated by the YTH domain containing protein Pho92/Mrb1 (Varier et al, 2022).…”