Piwi regulates the usage of alternative transcription start sites in the Drosophila ovary

Alternative transcription initiation, which refers to the transcription of a gene from different transcription start sites (TSSs), is prevalent in mammalian systems and has important biological functions. Although transcriptional regulation has been extensively studied, the mechanism that selects one TSS over others in a gene remains elusive. We discovered Piwi, an RNA-binding protein, regulated TSS usage of genes and identified 87 genes with significantly altered TSS usage (ATU) in piwi-deficient Drosophila ovaries using the cap-analysis gene expression sequencing (CAGE-seq) method. This regulation occurs in both germline and somatic cells in ovaries as well as in cultured ovarian somatic cells (OSCs). Correspondingly, RNA Pol II initiation and elongation at TSSs of ATU genes were affected in germline-piwi-knockdown ovaries and piwi-knockdown OSCs. We identified a FACT complex component, Ssrp, which is essential for mRNA elongation, as a novel interactor of Piwi in the nucleus. Temporally controlled knockdown of ssrp affected TSS usage of ATU genes whereas overexpression of ssrp partially rescued the TSS usage of ATU genes in piwi mutant ovaries. Thus, Piwi may interact with Ssrp to regulate TSS usage in Drosophila ovaries by affecting Pol II initiation and elongation. Overall design: To investigate the role of Piwi on transcription in germ cells, we conducted RNA-seq experiments using total RNA extracted from mature ovaries of germline-gfp-knockdown and germline-piwi-knockdown flies.