Project Summary Reversible chemical modifications of mRNAs have recently been recognized as a major regulatory mechanism of gene expression. Among >100 various RAN chemical modifications identified so far, N6- methyladenosine (m6A) represents the most abundant one with ~3-5 m6A sites per mRNA in eukaryotic transcriptomes. Our recent work (PNAS, 2017, 115:E325) has demonstrated that ALKBH5 acts as a m6A eraser, and m6A serves as a signal for alternative splicing in the nucleus of spermatocytes and round spermatids, and for degradation in the cytoplasm of elongating and elongated spermatids. The scientific premise that proper epitranscriptomic regulation (e.g., m6A) is essential for successful spermatogenesis and male fertility prompted us to embark on investigations aiming to understand how m6A levels are controlled in developing male germ cells and what role this specific chemical modification of mRNA plays in the regulation of spermatogenesis. Based upon our published and preliminary data, we hypothesize that ALKBH4, a homolog of ALKBH5, regulates mRNA m6A levels in spermatogenic cells either as a novel eraser or a co-factor of ALKBH5. To test this hypothesis, we will first determine whether ALKBH4 functions as a novel RNA m6A demethylase or as a co-factor for ALKBH5 using biochemical analyses in vitro (Aim1). The in vitro findings will then be validated using knockout mouse models in vivo and the physiological role of ALKBH4 in the regulation of spermatogenesis will also be determined (Aim2). The proposed project will help us gain insights into the epitranscriptomic regulation of spermatogenesis and the knowledge gained would help us discover the underlying causes of poor sperm quality and male infertility.