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 across metazoans and has important biological functions. Although transcriptional regulation has been extensively studied, the mechanism that selects one TSS over others within a gene remains elusive. Using the Cap Analysis of Gene Expression sequencing (CAGE-seq) method, we discovered that Piwi, an RNA-binding protein, regulates TSS usage in at least 87 genes. In piwi-deficient Drosophila ovaries, these genes displayed significantly altered TSS usage (ATU). The regulation of TSS usage occurred in both germline and somatic cells in ovaries, as well as in cultured ovarian somatic cells (OSCs). Correspondingly, RNA Polymerase II (Pol II) initiation and elongation at the TSSs of ATU genes were affected in germline-piwi-knockdown ovaries and piwi-knockdown OSCs. Furthermore, we identified a Facilitates Chromatin Transcription (FACT) complex component, Ssrp, that is essential for mRNA elongation, as a novel interactor of Piwi in the nucleus. Temporally controlled knockdown of ssrp affected TSS usage in 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.