Cutoff Suppresses RNA Polymerase II Termination to Ensure Expression of piRNA Precursors

Abstract

Small non-coding RNAs called piRNAs serve as guides for an adaptable immune system that represses transposable elements in germ cells of Metazoa. In Drosophila the RDC complex, composed of Rhino, Deadlock and Cutoff (Cuff) binds chromatin of dual-strand piRNA clusters, special genomic regions, which encode piRNA precursors. The RDC complex is required for transcription of piRNA precursors, though the mechanism by which it licenses transcription remained unknown. Here we show that Cuff prevents premature termination of RNA polymerase II. Cuff prevents cleavage of nascent RNA at poly(A) sites by interfering with recruitment of the cleavage and polyadenylation specificity factor (CPSF) complex. Cuff also protects processed transcripts from degradation by the exonuclease Rat1. Our work reveals a conceptually different mechanism of transcriptional enhancement. In contrast to other factors that regulate termination by binding to specific signals on nascent RNA, the RDC complex inhibits termination in a chromatin-dependent and sequence-independent manner.

Co-dependent Assembly of Drosophila piRNA Precursor Complexes and piRNA Cluster Heterochromatin

“…The Drosophila HP1 homolog Rhi anchors a chromatin complex that promotes cluster transcription (Andersen et al, 2017;Mohn et al, 2014), suppresses clustertranscript splicing (Zhang et al, 2014) and cluster transcript polyadenylation (Chen et al, 2016), and is essential to germline piRNA biogenesis (Klattenhoff et al, 2009; Mohn et al, 2014; Zhang et al, 2014). To determine if Rhi is required for cluster transcript binding to UAP56 and THO, we performed RIP sequencing (RIP-seq) with Thoc5GFP and UAP56venus in rhi 2/KG mutant ovaries.…”

“…Rhi interacts with the linker protein Del and the DXO homolog Cuff, forming the RDC complex, which promotes cluster transcription and suppresses cluster transcript splicing and polyadenylation (Chen et al, 2016; Mohn et al, 2014; Parhad et al, 2017; Zhang et al, 2014). A number of observations suggest that Rhi, functioning through Cuff, may indirectly promote UAP56 binding to cluster transcripts.…”

“…The resulting “non-canonical” transcripts are capped, and cap binding by the nuclear cap binding complex (CBC) promotes efficient splicing and polyadenylation (Proudfoot et al, 2002). However, cluster transcripts are not spliced or polyadenylated (Chen et al, 2016; Zhang et al, 2014). Cuff is a homolog of the decapping enzyme DXO, but the catalytic pocket is not conserved, and residues that interact with the RNA backbone are retained (Zhang et al, 2014).…”

“…In the Drosophila female germline, piRNAs are processed from RNA polymerase II (RNA Pol II) transcripts of discrete genomic domains called piRNA clusters, composed of nested transposon fragments, which provide an archive of invading transposon sequences (Andersen et al, 2017; Bergman et al, 2006; Brennecke et al, 2007; Chen et al, 2016; Mohn et al, 2014; Zhang et al, 2012a, 2014). Transposition of an invading mobile element into a cluster is proposed to trigger adaptation, and the system must therefore have ability to process any inserted sequence into piRNAs.…”

“…These three factors are co-dependent for localization to cluster heterochromatin, drive non-canonical transcription of piRNA clusters from both genomic strands, suppress cluster transcript splicing and polyadenylation, and promote piRNA biogenesis (Andersen et al, 2017; Chen et al, 2016; Klattenhoff et al, 2009; Mohn et al, 2014; Pane et al, 2011; Parhad et al, 2017; Zhang et al, 2014). Like the founding member of the HP1 family, HP1a, Rhi binds to trimethylated lysine 9 of histone 3 (H3K9me3) through its C-terminal chromo domain (Le Thomas et al, 2014; Mohn et al, 2014; Yu et al, 2015).…”