Biology subjects

Ensinck, I.

Publications and source records attributed to Ensinck, I..

3 recordsLinked to original sources

bioRxiv2023

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

N6-methyladenosine (m6A), the most abundant mRNA modification, is deposited in mammals/insects/plants by m6A methyltransferase complexes (MTC) comprising a catalytic subunit and at least five additional proteins. The yeast MTC is critical for meiosis and was known to comprise three proteins, of which two were conserved. We uncover three novel MTC components (Kar4/Ygl036w-Vir1/Dyn2). All MTC subunits, except for Dyn2, are essential for m6A deposition and have corresponding mammalian MTC orthologs. Unlike the mammalian bipartite MTC, the yeast MTC is unipartite, yet multifunctional. The mRNA interacting module, comprising Ime4, Mum2, Vir1, and Kar4, exerts the MTCs m6A-independent function, while Slz1 enables the MTC catalytic function in m6A deposition. Both functions are critical for meiotic progression. Kar4 also has a mechanistically separate role from the MTC during mating. The yeast MTC constituents play distinguishable m6A-dependent, MTC-dependent and MTC-independent functions, highlighting their complexity and paving the path towards dissecting multi-layered MTC functions in mammals.

molecular biology↗

bioRxiv2022

m6A-ELISA, a simple method for quantifying N6-methyladenosine from mRNA populations

N6-methyladenosine (m6A) is a widely studied and abundant RNA modification. The m6A mark regulates the fate of RNAs in various ways, which in turn, drives changes in cell physiology, development, and disease pathology. Over the last decade, numerous methods have been developed to map and quantify m6A sites genomewide through deep sequencing. Alternatively, m6A levels can be quantified from a population of RNAs using techniques such as liquid chromatography-mass spectrometry or thin layer chromatography. However, many methods for quantifying m6A levels involve extensive protocols and specialized data analysis, and often only a few samples can be handled in a single experiment. Here, we developed a simple method for determining m6A levels in mRNA populations from various sources based on enzyme-linked immunosorbent-based assay (m6A-ELISA). We have optimized various steps of m6A-ELISA such as sample preparation and the background signal resulting from the primary antibody. We validated the method using mRNA populations from budding yeast and mouse embryonic stem cells. The full protocol takes less than a day, requiring only 25 ng of mRNA. The m6A-ELISA protocol is therefore quick, cost-effective, and scalable, making it a valuable tool for determining relative m6A levels in samples from various sources that could be adapted to detect other mRNA modifications.

biochemistry↗

bioRxiv2022

m6A reader Pho92 is recruited co-transcriptionally and couples translation efficacy to mRNA decay to promote meiotic fitness in yeast

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 3end 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.

biochemistry↗