Revealing RNA poly(A) tail dynamics during spermiogenesis and its function to support male fertility [dRNA-seq_busulfan]

To investigate the poly(A) polymerase activity of TENT5C in testicular somatic cells, we enriched the testis of wild-type and TENT5cdcat/dcat mice with interstitial cells by depleting germ cells with busulfan. Sequencing of busulfan treated testis revealed Insl3 transcripts with poly(A) tails almost exclusively accumulating at ~180-nts in wild-type samples. In Tent5c mutants, Insl3 transcript show gradual deadenylation down to ~60-nt with a significant depletion of the ~180-nt long tails. A significant decrease in Insl3 transcripts was also observed in TENT5cdcat/dcat busulfan treated testis compared to wild-type. These results indicate that TENT5C poly(A) polymerase is required to maintain or readenylate Insl3 poly(A) tails up to ~180-nts in interstitial cells. The shortening of the poly(A) tail may lead to transcripts decay. Overall design: Tent5c wt/wt or Tent5c dcat/dcat adult male mice were injected with a single dose of busulfan 20mg/kg or the corresponding vehicle. Testis were harvested 4-weeks after injection to allow for an adequate depletion of germ cells. RNA was isolated from total testis. Each sample is representative of individual mice - 2 replicates per condition were considered for analysis. Nanopore technologies were used to sequence the testicular transcriptome and determine mRNA poly(A) tail length.