Transposable element dynamics and PIWI regulation impacts lncRNA and gene expression diversity in Drosophila ovarian cell cultures

Abstract

Piwi proteins and Piwi-interacting RNAs (piRNAs) repress transposable elements (TEs) from mobilizing in gonadal cells. To determine the spectrum of piRNA-regulated targets that may extend beyond TEs, we conducted a genome-wide survey for transcripts associated with PIWI and for transcripts affected by PIWI knockdown in Drosophila ovarian somatic sheet (OSS) cells, a follicle cell line expressing the Piwi pathway. Despite the immense sequence diversity among OSS cell piRNAs, our analysis indicates that TE transcripts are the major transcripts associated with and directly regulated by PIWI. However, several coding genes were indirectly regulated by PIWI via an adjacent de novo TE insertion that generated a nascent TE transcript. Interestingly, we noticed that PIWI-regulated genes in OSS cells greatly differed from genes affected in a related follicle cell culture, ovarian somatic cells (OSCs). Therefore, we characterized the distinct genomic TE insertions across four OSS and OSC lines and discovered dynamic TE landscapes in gonadal cultures that were defined by a subset of active TEs. Particular de novo TEs appeared to stimulate the expression of novel candidate long noncoding RNAs (lncRNAs) in a cell lineage-specific manner, and some of these TE-associated lncRNAs were associated with PIWI and overlapped PIWI-regulated genes. Our analyses of OSCs and OSS cells demonstrate that despite having a Piwi pathway to suppress endogenous mobile elements, gonadal cell TE landscapes can still dramatically change and create transcriptome diversity.

Drosophila PAF1 Modulates PIWI/piRNA Silencing Capacity

“…Overall, mature TE RNAs were more strongly up-regulated than nascent RNAs after siPIWI knockdown (Fig. 3A), consistent with previous observations that PIWI uses transcriptional and post-transcriptional mechanisms [7, 26, 38]. In siPAF1 knockdowns, TE nascent RNAs increased while TE mature RNAs decreased (Fig.…”

“…Although several TEs were up-regulated at both nascent and mature RNA levels, some TEs, like ZAM, copia, and springer , which have increased genomic copy numbers in OSS cells [38], were mainly up-regulated at the mature RNA level after siPIWI knockdown (Figure 3A, Figure S3). Overall, mature TE RNAs were more strongly up-regulated than nascent RNAs after siPIWI knockdown (Fig.…”

“…PIWI-silencing affects expression of genes near TE insertions [7, 10, 38]. About ~9% of genes near TEs were up-regulated at nascent RNA levels after siPIWI knockdown, whereas ~35% of genes were up-regulated at mature RNA levels, highlighting post-transcriptional regulation (Fig.…”

“…OSS cells also express TE-linked lncRNAs that can interact with PIWI independently of piRNAs [38]. TE-linked lncRNA expression was increased after PIWI or PAF1 knockdown, but was re-balanced when PIWI and PAF1 were reduced together (Fig.…”

The capacity of target silencing by Drosophila PIWI and piRNAs

“…In OSS and OSC cells, PIWI operates at the nascent RNA level to silence endogenous TEs and genes adjacent to a transcriptionally active TE (Sienski et al 2012;Sytnikova et al 2014). To determine whether the PIWI-dependent regulation of our reporter transgenes was also due to TGS or PTGS, we inserted the flam element into the 5 ′ UTR of the luciferase reporter, either juxtaposed to the transcriptional start site (TSS) or within a small and efficiently spliced intron in the 5 ′ UTR that enhances the translation of the luciferase (Supplemental Fig.…”