What can budding yeast teach us about the role of mRNA’s fifth base?

DNA, RNA and protein can all be modified following synthesis. These modifications have been relatively intensively studied in DNA and proteins, and have been found to dramatically impact the properties and the biological regulation of the molecules harboring them. However, far less is known about modifications on mRNA. N6-methyladenine (m6A) is a highly ubiquitous base modification occurring on mammalian mRNA. Discovered almost four decades ago, this modification is present within over 50% of mammalian transcripts, typically near stop codons and within very long internal exons. Orthologs of the RNA-based N6-adenosyl methyltransferases (MTases) that catalyze this modification are present in almost all eukaryotes, and their depletion or disruption causes lethality in metazoans and severe developmental defects in plants, thus underscoring their essentiality.

HFSP Long-Term Fellow Schraga Schwartz and colleagues
authored on Fri, 24 January 2014

Significant technical and experimental limitations have hindered the study of m6A modifications. First, since m6A neither changes the base-pairing properties nor inhibits reverse transcription, this imposes substantial difficulties in identifying methylated sites. The best maps generated to date, based on immunoprecipitation using antibodies against m6A, were at a resolution of ~24nt, making it difficult to pinpoint the precise methylated base. Second, experimental depletion of the methylation complex in mammals results in apoptosis, rendering it difficult to dissect the functional role of methylation. Third, the mammalian methylation landscape appears to be mostly static across mammalian cell types, tissues, and stimuli, limiting our ability to elucidate how methylations emerge. To overcome these difficulties, we examined mRNA methylation in the budding yeast, Saccharomyces cerevisiae. In yeast, mRNA methylation occurs exclusively during meiosis, providing a unique opportunity to dissect its dynamics and regulation. Moreover, the methyltransferases involved in mediating methylation are not essential for viability, allowing experimental exploitation of strains lacking these modifications.

By optimizing the mapping protocol, and exploiting yeast strains lacking the ability to methylate mRNA, we were able to generate near single nucleotide transcriptome-wide maps of m6A sites in meiotic yeast transcripts, and identified 1,308 putatively methylated sites within 1,183 different transcripts. We validated 8/8 methylation sites in different genes with direct genetic analysis, and showed that methylated sites are significantly conserved in a related species, Saccharomyces mikatae. By analyzing the sequence-based characteristics of methylation sites, we were able to build a model that predicts methylated sites directly from sequence. Examination of m6A sites along a dense meiotic time course revealed that different sites display different methylation dynamics, which are regulated jointly in ‘cis’, via predictable ‘methylatability’ of each site, and ‘in trans’ through regulation of availability of core meiotic circuitry. We determined that the methyltransferase complex components localize to the yeast nucleolus, and this localization is essential for mRNA methylation. Finally, we identified a novel protein that specifically binds to methylated mRNA in yeast and moreover found that the entire machinery of methylation ‘readers’ and ‘writers’ has coevolved between yeast and humans, suggesting that the broad principles identified here are to a large extent relevant in the mammalian system.

Our data illuminates a conserved, dynamically regulated methylation program in yeast meiosis, and paves the way towards understanding the function of this ‘epitranscriptomic’ modification.

Reference

High-resolution mapping reveals a conserved, widespread, dynamic mRNA methylation program in yeast meiosis. Schraga Schwartz, Sudeep D. Agarwala, Maxwell R. Mumbach, Marko Jovanovic, Philipp Mertins, Alexander Shishkin, Yuval Tabach, Tarjei S. Mikkelsen, Rahul Satija, Gary Ruvkun, Steven A. Carr, Eric S. Lander, Gerald R. Fink, Aviv Regev. Cell Volume 155, Issue 6, 1409-1421, 21 November 2013.

Link to Cell article

Pubmed link