Poster abstracts
Poster number 70 submitted by Mohamed Rushdhi Mohamed Rauff
GSK3α plays a vital role in male fertility by regulating m6A levels in testis
Rushdhi Rauff (Department of Chemistry and Biochemistry, Kent State University), Souvik Dey (Department of Biological Sciences, Kent State University), Wesam Nofal (Department of Biological Sciences, Kent State University), Mustfa Kabi (Department of Biological Sciences, Kent State University), Srinivasan Vijayaraghavan (Department of Biological Sciences, Kent State University), Sanjaya Abeysirigunawardena (Department of Chemistry and Biochemistry, Kent State University)
Abstract:
N6-methyladenosine (m6A) is the most abundant nucleotide modification observed in eukaryotic mRNA. N6-methylation levels are dynamic in mRNA, where methylations are added and removed by proteins that are known as writers and erasers, respectively. FTO (Fat mass and obesity-associated protein) is one of the most well-studied methyl eraser proteins found in eukaryotes. It has been known that in stem cells, FTO is a substrate of GSK3 protein. GSK3 enzyme, a serine/threonine protein kinase, is ubiquitous and is expressed as two paralogs, GSK3α and GSK3β, encoded by two genes. The only phenotype of global deletion of Gsk3α is male infertility. Here we hypothesize that GSK3α plays a similar role in controlling m6A levels in testis by interacting with FTO. Our experiments show that FTO is localized to post-meiotic spermatogenic cells within the seminiferous tubules. There is no visible staining for FTO in Sertoli cells, spermatogonia, myoid, and Leydig cells in the interstitial spaces of the seminiferous tubules. Co-immunoprecipitation of FTO with GSK3α, but not with GSK3β was observed when immunoprecipitation was carried out for GSK3α and GSK3β followed by western blots using FTO primary antibodies. A marked increase in FTO expression was seen in day 20 testis up to day 40 adult testis; an expression pattern characteristic of proteins expressed in post-meiotic developing spermatocytes and spermatids. Using HPLC and ELISA-based methods, we observed that m6A levels are significantly lower in GSK3α KO mice compared to WT mice. Hence, our experimental findings illustrate that GSK3α plays a main role in male fertility by regulating m6A levels in testis.
Keywords: m6A, nucleotide modifications, epitranscriptomics