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) bind 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.

Fig. 1. Cuff deficiency eliminates the majority of transcripts from piRNA clusters

(A) Cuff depletion leads to diminished piRNA cluster transcription. Shown are profiles of piRNA, total cell and poly(A)-selected RNA-seq, chromatin RNA-seq, global nuclear run-on seq (GRO-seq), and RNA polymerase II ChIP-seq from ovaries of control and cuff depleted flies on the dual-strand piRNA cluster 38C. The distribution of predicted poly(A) signals on plus and minus genomic strands is shown in the poly(A) signal (PAS) tracks. Below is an expansion of the indicated region from which poly-adenylated transcripts detected by RT-PCR using oligo-dT primer are produced in cuff^wm25/qq37 flies. (B) Chromatin-associated RNA sequencing libraries are depleted of signal recognition particle (7SL) RNA. (C) Chromatin-associated RNAs are enriched in intronic sequences as measured by the ratio of intronic to exonic RPKM signals. Solid line indicates no difference between standard and chromatin RNA-seq. (D) piRNA cluster transcripts are enriched in chromatin-associated RNA. (E) Cuff suppresses primary transcripts from Cuff-depended piRNA producing loci. Heatmap shows signal change in Cuff-depleted versus control ovaries for small RNA, total RNA, chromatin-associated RNA and GRO-seq in genomic windows that produce piRNA in a Cuff-dependent manner. Intervals overlapping uni-strand piRNA clusters that are not affected by Cuff depletion are shown for comparison (Flam+20A). (F) Box plots show the distribution of piRNA, chromatin-associated RNA, total RNA and GRO signals in control and Cuff-depleted ovaries. Green boxes corresponds to all genomic regions that produce piRNA in Cuff-dependent manner; pink boxes correspond to windows overlapping uni-strand piRNA clusters (flamenco and 20A). See also Figure S1.

Fig. 2. Cuff suppresses transcription termination at canonical poly(A) sites

(A) Profiles of different libraries over the 42AB cluster which is further divided into thee regions, A, B and C. Below shown are the locations of two insertions of artificial MiMIC transposons, and the site of CRISPR-mediated deletion of promoters of the Pld gene, which flanks the 42AB cluster. The entire cluster is shown on Figure S2A. (B) Cuff depletion leads to reduced transcription of the 42AB cluster. Normalized densities of GRO-seq and chromatin RNA-seq signals are calculated for three regions shown in (A). (C) Depletion of Cuff has a different effect on two promoterless MiMIC insertions located at different positions in the 42AB cluster. Shown is the amount of MiMIC RNA in control and cuff-depleted (shCuff) ovaries as determined by RT-qPCR. Error bars show standard errors from three biological replicates. (D) Depletion of Cuff suppresses read-through transcription downstream of the canonical poly(A) site of the M8627 insertion. Amounts of RNA upstream (GFP1-4) and downstream (ppA) of the poly(A) site were quantified by strand-specific RT-qPCR and the ratios of read-through (ppA/GFP1-4) transcripts were plotted. RT primers and qPCR amplicons are shown in panel C. Error bars show standard errors from three biological replicates. See also Figure S2B.

Fig. 3. Exposure of target locus to homologous piRNA enhances read-through transcription

(A) Exposure to maternally inherited homologous piRNAs activates piRNA biogenesis. The insertion of the P{lArB} construct in a subtelomeric piRNA cluster in P1152 strain produces abundant piRNAs that target homologous loci, the lacZ-containing BC69 transgene and the endogenous hsp70 locus, in trans. piRNA biogenesis from target loci is activated if inducer is inherited from the mother (‘activated’ progeny), but not in control flies produced by the reciprocal cross. The right panel shows the density of piRNAs at the endogenous hsp70 locus in the ovaries of activated and control progenies compared to 42AB cluster. (B) Rhi and Cuff associate with target loci exposed to homologous piRNA. Binding of Rhi and Cuff to chromatin of the BC69 transgene (T1) and the hsp70 locus (H1) was measured in ovaries of activated and control progenies by ChIP-qPCR using amplicons shown in (A). Error bars represent standard deviations of six technical replicates. (C) Activation of primary piRNA biogenesis on the hsp70 locus is associated with increased read-through transcription as measured by strand-specific RT-qPCR using primers shown in panel A. Error bars represent the standard deviations of three biological replicates. See also Figure S3.

Fig. 4. Tethering of Cuff to a heterologous mRNA reporter suppresses termination and poly(A) site cleavage

(A) Schematic diagram of the reporter used to study the effect of Cuff recruitment to RNA. Cuff fused to the λN peptide, which binds BoxB hairpins in reporter RNA, was co-expressed with the reporter in ovaries. (B) Cuff tethering leads to increased reporter transcript levels. λN-Cuff or control λN-GFP were co-expressed with the reporter in fly ovaries and RNA levels from three regions shown on (A) were quantified by RT-qPCR. Error bars show standard errors of four biological replicates. (C) Tethering of Cuff increases read-through transcription of the reporter pre-mRNA as measured by the fraction of read-through transcripts relative to total reporter RNA. Error bars are standard errors from four biological replicates. P values were calculated by t-test. See also Figure S4. (D) Tethering of Cuff suppresses Pol II termination as measured by Pol II ChIP-qPCR downstream of the poly(A) signals (fragments 4 and 5 on panel A). Error bars show standard errors of two biological replicates. (E) Tethering of Cuff inhibits cleavage at poly(A) sites. Shown is the fraction of unprocessed transcripts calculated as described on Figure S5. Error bars are standard errors from four biological replicates. P was calculated by t-test. (F) Unprocessed reporter transcripts that span the poly(A) site are detected by RT-PCR upon λN-Cuff tethering (amplified regions are shown in panel A). (G) Tethering of Cuff suppresses binding of CPSF complex as measured by Cpsf73 ChIP-qPCR on different portions of the reporter shown on (A). Error bars show standard errors of four biological replicates. (H) The majority of read-through transcripts formed upon Cuff tethering are cleaved near poly(A) sites. The fractions of cleaved and unprocessed RNA in two biological replicas were determined using standard curves shown on Figure S4 and S5.

Fig. 5. Cuff is required for Rhi-mediated suppression of splicing

(A) Schematic diagram of the intron-containing reporter used to study the effect of Cuff recruitment on splicing. (B) Cuff is required for Rhi-mediated suppression of splicing. The splicing efficiency was measured by the ratio of spliced to unspliced transcripts.

Fig. 6. Cuff stabilizes non-capped transcripts formed by cleavage at poly(A) sites

(A) Anti-trimethylguanosine antibody specifically precipitates fully capped RNA. Radiolabeled RNAs with different groups at the 5’ end were precipitated with anti-trimethylguanosine antibody and bound (IP) and unbound fractions resolved on PAAG. (B) Tethering of Cuff increases the fraction of non-capped read-through reporter transcripts. Capped RNA was immunoprecipitated from total ovarian RNA of flies that express the 4BoxB reporter shown on Fig 4A. Error bars show standard errors from six biological replicates. P values were calculated by t-test. (C) Anti-CBP80 antibody specifically binds to Drosophila CBP80 protein. See also Figure S6. (D) Tethering of Cuff increases association of CBP80 with reporter RNA. Ovarian lysates from flies expressing the 4BoxB reporter were used for RNA immunoprecipitation (RIP) with anti-CBP80 antibody. Error bars are standard deviations from two biological replicates. (E) Tethering of Cuff increases association of CBP80 with chromatin of the reporter locus. ChIP using paraformaldehyde (PFA) or ethylene glycol bis(succinimidyl succinate) (EGS) crosslinkers were used to determine association of CBP80 with several regions of the reporter shown in Fig. 4A. Error bars show standard errors from two technical replicates.

Fig. 7. Depletion of Rat1 exonuclease suppresses effects of the cuff mutation

(A) Drosophila Rat1 and dRai1 have exonuclease activity toward 5’-monophosphate RNA. After incubation with the purified proteins radiolabeled RNA was resolved on a denaturing PAGE. NC: no protein control. (B) dRai1 but not Cuff has pyrophosphohydrolase activity. See Suppl. methods for details. (C) Drosophila dRai1 and Cuff physically interacts with Rat1 as measured by Co-IP/Western of tagged proteins. (D) Cuff and Rat1 are localize in nuclei. Immunostaining of GFP-tagged Cuff and BioTAP-tagged Rat1 proteins in ovaries. DAPI staining was used to locate nuclei. (E) Inhibition of expression of rat1 increases the levels of read-through transcripts downstream of the canonical poly(A) site as measured by RT-qPCR on reporter shown in Fig. 4A. Error bars show standard errors for three biological replicates. (F-G) Inhibition of rat1 in germline suppresses the phenotype of cuff^wm25/qq37 mutation. The plots show the change in expression of piRNA cluster transcripts (F) and transposable elements (G) measured by RT-qPCR in ovaries of flies of specified genotype. Error bars show standard error among four (F) or three (G) biological replicates. P-values are calculated by t-test.