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.
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
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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Further reading
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- Biochemistry and Chemical Biology
Escherichia coli ClpB and Saccharomyces cerevisiae Hsp104 are AAA+ motor proteins essential for proteome maintenance and thermal tolerance. ClpB and Hsp104 have been proposed to extract a polypeptide from an aggregate and processively translocate the chain through the axial channel of its hexameric ring structure. However, the mechanism of translocation and if this reaction is processive remains disputed. We reported that Hsp104 and ClpB are non-processive on unfolded model substrates. Others have reported that ClpB is able to processively translocate a mechanically unfolded polypeptide chain at rates over 240 amino acids (aa) per second. Here, we report the development of a single turnover stopped-flow fluorescence strategy that reports on processive protein unfolding catalyzed by ClpB. We show that when translocation catalyzed by ClpB is challenged by stably folded protein structure, the motor enzymatically unfolds the substrate at a rate of ~0.9 aa s−1 with a kinetic step-size of ~60 amino acids at sub-saturating [ATP]. We reconcile the apparent controversy by defining enzyme catalyzed protein unfolding and translocation as two distinct reactions with different mechanisms of action. We propose a model where slow unfolding followed by fast translocation represents an important mechanistic feature that allows the motor to rapidly translocate up to the next folded region or rapidly dissociate if no additional fold is encountered.
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- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
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