Characterizing Alternative Polyadenylation in Male Germ Cells Using Poly(A)-seq

Post-transcriptional processing of mRNA subsequently determines its longevity, transport, and gene expression. Alternative polyadenylation (APA), one form of post-transcriptional processing, is the use of a variant polyadenylation signal and polyA-site for transcript cleavage and addition of adenine residues. The occurrence of this phenomenon in the 3’-untranslated region (3’UTR) can lead to transcript isoforms differing in 3’UTR length and as a result, alter the downstream cis-regulatory elements in use. Transcript modification at the 3’-end in alternative polyadenylation has been shown to foster a number of diseases in altering what would otherwise be normal protein expression and is furthermore emerging as a driver of spermatogenesis. To date, the global mechanisms that control the post-transcriptional processing in male germ cells remain unknown. PolyA-seq is a strand-specific, quantitative method for the high-throughput sequencing of 3’-ends of transcripts post-transcriptionally modified in polyadenylation. It has the ability to accurately and globally map polyA-sites. To study the molecular regulation of sperm production, an in-depth bioinformatics analysis was performed on available PolyA-seq data to identify male germ cell transcripts that uniquely use alternative polyadenylation, a novel method was developed to isolate and purify male germ cells from testicular tissue, and libraries were prepared for PolyA-seq from isolated male germ cells. Findings show no significant global difference in polyadenylation signal use between testicular and liver tissues when the same polyadenylation site is compared for human PolyA-seq data. Further annotation suggests a conservation in polyadenylation signal and polyA-site use across tissue types in the same species. Transcripts in the liver were more likely to use the canonical polyadenylation signal in comparison to those in the testis, lending further evidence of increased variant polyadenylation signal use in male germ cells attributed to alternative polyadenylation. Moreover, manual identification of known alternatively polyadenylated transcripts in testis from mice suggests that PolyA-seq is a reliable method for transcriptome characterization. Isolation and purification of male germ cells was successful using DRAQ5 nuclear stain. Using the verified isolated male germ cells, PolyA-seq libraries were generated. Comparison of different polyadenylation sites for the same transcripts between testis and liver PolyA-Seq still needs to be conducted. Also, increasing yields of polyA+ RNA for PolyA-Seq library prep will facilitate successful sequencing of 3’-ends. Further investigation of male germ cell-specific transcripts associated with alternative polyadenylation will lead to an improved understanding of molecular regulation involved in spermatogenesis and factors that cause male infertility.

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