Idiopathic male infertility is a rising global concern characterised by ejaculatory defects, absence or low sperm count with abnormal morphology, and poor sperm motility. Deciphering the aetiology of male infertility requires a fundamental understanding of multiple spermatogenic events, including sperm maturation. Glycogen synthase kinase 3 paralog-α (GSK3α) plays a critical role in sperm maturation, specifically during epididymal motility, capacitation and hyperactivation. Methylation modulates mRNA transport, stability, turnover, and translational efficiency to meet cellular requirements. The mRNA demethylase, fat mass and obesity-associated protein (FTO), is a target for phosphorylation by GSK3α, suggesting the potential role of this enzyme in male fertility. This study focuses on the high-affinity spatiotemporal interaction between FTO and GSK3α to delineate the post-transcriptional modifications in the murine testis. Expression of Gsk3a and Fto increases temporally starting with day 18-20 postpartum testis, coinciding with meiosis I and gradually peaks by day 25-34 with the formation of spermatids and completion of spermatogenesis. Co-immunoprecipitation of GSK3α and GSK3β with FTO using respective antibodies and super-resolution microscopy shows a preferential interaction of GSK3α with FTO. Moreover, Gsk3a knockout mice showed significantly low m6A levels in testis, presumably due to enhanced FTO activity. In silico protein-protein docking and molecular dynamics analysis demonstrated an energetically favourable, consensus phosphorylation motif-dependent high-affinity interaction between FTO and GSK3α, further validating our observation. A specific missense mutation (Cys326 > Ser) permitted an additional GSK3-phosphorylation site in FTO, leading to teratozoospermia in a patient. Collectively, this study affirms GSK3α as a spatiotemporal regulator of FTO function inside the mammalian testis.
Neha Choudhari, B. Dehury, Rounak Roy et al.· The FEBS Journal· 0 citations
N6-methyladenosine profiles of mRNA transcripts regulate their translocation from the nucleus to the cytosol, stability, and translational efficiency; hence, they have been implicated in gene expression and disease progression. The m6A-methylation is widely associated with various cancers and neurological, cardiovascular, and developmental disorders, which demand early diagnosis. A robust m6A-motif prediction is necessary to enable us to identify the regulatory nucleic acid sequences that determine mRNA fate in normal and diseased conditions. We have developed a transcript-aware computational pipeline, termed m6AFunctional Index in Transcription (m6A-FINDiT), that can identify potential m6A sites on mRNA transcripts, considering molecular intricacies associated with their secondary structure. This tool can separately identify m6A motifs within the coding sequences as well as in non-translatable regions, i.e., 5’UTR and 3’UTR, of mRNA transcripts. Parallelly, another technique was developed that quantifies specific m6A methylation motifs through a probe-based ELISA process, MAQ-G. This second method successfully validated the N⁶-methyladenosine motifs predicted by the initially developed motif-finder program. This integrated m6A-FINDiT and MAQ-G, coupled with a real-time qPCR assay, could correlate the methylation profiles of N6-methyladenosine motifs with the expression and stability contours of a gene. To establish the physiological implications of these techniques, we chose three tumour-suppressor genes, viz., IRF8, RB1, and TP53 mRNA transcripts, which may undergo m6A methylation at certain DRACH motifs. The m6A-FINDiT pipeline could successfully predict the specific m6A motifs, and the MAQ-G confirmed the methylation profile of the latter. These duo techniques hold potential for use in clinical settings for early cancer detection.
Neha Choudhari, H. Srinivas, Praneeth Sai Tadepalli et al.· Molecular Biology Reports· 0 citations
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