N6-methyladenosine (m6A) reader Pho92 is recruited co-transcriptionally and couples translation to mRNA decay to promote meiotic fitness in yeast
- The Francis Crick Institute, United Kingdom
- Radboud University Nijmegen, Netherlands
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.
Data availability
The miCLIP, iCLIP and RNA-seq RAW and processed data are available to review GEO accession GSE193561:https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE193561
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High-resolution view of the yeast meiotic program revealed by ribosome profilingNCBI Gene Expression Omnibus, GSE34082.
Article and author information
Author details
Folkert Jacobus van Werven
Funding
Wellcome Trust (FC001203)
- Radhika A Varier
- Theodora Sideri
- Zornitsa Manova
- Alice Rossi
- Imke Ensinck
- Folkert Jacobus van Werven
Cancer Research UK (FC001203)
- Radhika A Varier
- Theodora Sideri
- Zornitsa Manova
- Alice Rossi
- Imke Ensinck
- Folkert Jacobus van Werven
Medical Research Council (FC001203)
- Radhika A Varier
- Theodora Sideri
- Zornitsa Manova
- Alice Rossi
- Imke Ensinck
- Folkert Jacobus van Werven
Wellcome Trust (FC010110)
- Charlotte Capitanchik
- Nicholas M Luscombe
Cancer Research UK (FC010110)
- Charlotte Capitanchik
- Nicholas M Luscombe
Medical Research Council (FC010110)
- Charlotte Capitanchik
- Nicholas M Luscombe
Medical Research Council (FC001134)
- Enrica Calvani Enrica.Calvani
- Markus Ralser
Cancer Research UK (FC001134)
- Enrica Calvani Enrica.Calvani
- Markus Ralser
Dutch Cancer Society
- Raghu R Edupuganti
- Michiel Vermeulen
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, Varier et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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N6-methyladenosine (m6A) reader Pho92 is recruited co-transcriptionally and couples translation to mRNA decay to promote meiotic fitness in yeast
eLife 11:e84034.
https://doi.org/10.7554/eLife.84034
Further reading
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- Biochemistry and Chemical Biology
N 6,2’-O-dimethyladenosine (m6Am) is a modified nucleotide located at the first transcribed position in mRNA and snRNA that is essential for diverse physiological processes. m6Am mapping methods assume each gene uses a single start nucleotide. However, gene transcription usually involves multiple start sites, generating numerous 5’ isoforms. Thus, gene-level annotations cannot capture the diversity of m6Am modification in the transcriptome. Here, we describe CROWN-seq, which simultaneously identifies transcription-start nucleotides and quantifies m6Am stoichiometry for each 5’ isoform that initiates with adenosine. Using CROWN-seq, we map the m6Am landscape in nine human cell lines. Our findings reveal that m6Am is nearly always a high stoichiometry modification, with only a small subset of cellular mRNAs showing lower m6Am stoichiometry. We find that m6Am is associated with increased transcript expression and provide evidence that m6Am may be linked to transcription initiation associated with specific promoter sequences and initiation mechanisms. These data suggest a potential new function for m6Am in influencing transcription.
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- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.
