Drosophila PAF1 Modulates PIWI/piRNA Silencing Capacity

Abstract

To test the directness of factors in initiating PIWI-directed gene silencing, we employed a Piwi-interacting RNA (piRNA)-targeted reporter assay in Drosophila ovary somatic sheet (OSS) cells [1]. This assay confirmed direct silencing roles for piRNA biogenesis factors and PIWI-associated factors [2-12] but suggested that chromatin-modifying proteins may act downstream of the initial silencing event. Our data also revealed that RNA-polymerase-II-associated proteins like PAF1 and RTF1 antagonize PIWI-directed silencing. PAF1 knockdown enhances PIWI silencing of reporters when piRNAs target the transcript region proximal to the promoter. Loss of PAF1 suppresses endogenous transposable element (TE) transcript maturation, whereas a subset of gene transcripts and long-non-coding RNAs adjacent to TE insertions are affected by PAF1 knockdown in a similar fashion to piRNA-targeted reporters. Additionally, transcription activation at specific TEs and TE-adjacent loci during PIWI knockdown is suppressed when PIWI and PAF1 levels are both reduced. Our study suggests a mechanistic conservation between fission yeast PAF1 repressing AGO1/small interfering RNA (siRNA)-directed silencing [13, 14] and Drosophila PAF1 opposing PIWI/piRNA-directed silencing.

Keywords: Drosophila; PAF1; Piwi; gene silencing; piRNA.

Figure 1. Reporter assay to test factors impacting initiation of PIWI-directed silencing

(A) Left, design of the piRNA-targeted reporter assay in OSS cells. Right, a 2.4 kb piRNA-targeted region from the Flamenco (Flam) locus inserted in “Sense” (same orientation as piRNAs) or “Antisense” configurations into the 5'-intron or 3'UTR of the Renilla luciferase plasmid. (B) Normalization procedure measures a factor’s impact on PIWI-directed silencing. Left, representative values of Renilla luciferase to firefly luciferase. Antisense reporter (dark blue) is repressed by PIWI/piRNAs in the siGFP negative control, but de-repressed upon PIWI knockdown (KD, by siPIWI siRNA). Right, full normalization of siPIWI KD changes against siGFP control. (C) Assay of known PIWI/piRNA pathway factors shows greater reporter de-repression when the Flam element is in the 5'-intron compared to the 3'UTR. (D) Knockdown of chromatin associated factors do not display reporter de-repression. (E) Assay of RNA Polymerase II-associated factors show loss of PAF1 and RTF1 enhance silencing of 5' intron piRNA-targeted reporters. Standard deviation of biological triplicates with significant differences to siGFP control; *=p<0.05, **=p<0.01, t-test. See also Figures S1 and S2.

Figure 2. PAF1 modulation of piRNA-targeted reporters

(A) Left, piRNA profiles for a Flam 600bp element and a Tj-3'UTR 300bp element. Right, positions of Sense and Antisense piRNA-target segments inserted into 5'UTR, 5'intron, 3'intron, or 3'UTR reporter regions. Assay results for Flam-600bp-element (B) and Tj-3'UTR-300bp-element (C) during siPIWI and siPAF1 treatment. (D) Assay results of factors impacting transcription termination (CSTF64), elongation (DRE4/SPT16), and RNA Pol II pausing (CDK9/pTEFb). Standard deviation of biological triplicates with significant differences to siGFP control; *=p<0.05, **=p<0.01, t-test.

Figure 3. OSS cell transcriptomes after PAF1 knockdown display greater repression of TE transcripts

(A) Boxplot of TE expression changes with minimum and maximum whiskers, and **=p<0.01, ***=p<0.001; Wilcoxon rank sum test. (B) Coverage plots for nascent (top tracks) and mature RNA (bottom tracks) reads for TE consensus sequences, with small RNAs on the lowest track. Additional TE consensus tracks are in Fig. S3B. (C) Boxplot of nascent and mature RNAs from expressed genes grouped by regulation and TE features, with the number of nascent and mature RNA genes in parentheses, respectively. Whiskers represent minimum and maximum values, and ***=p<0.001; Wilcoxon rank sum test. (D) Coverage plots for genes upregulated by siPIWI but downregulated by siPAF1, either at the nascent RNA (Crb), mature RNA (Sbb), or at both levels (CG34393 and CG6441). Full gallery is in Fig. S3H. (E) Boxplot of TE-linked lncRNAs. Whiskers represent minimum and maximum values, and *=p<0.05, Wilcoxon rank sum test. (F) Coverage plots of lncRNAs upregulated by siPIWI but downregulated by siPAF1. (G) Transcripts upregulated by siPIWI-knockdown but cannot be called lncRNAs because of the same strand polarity in coverage across protein-coding genes. Dashed line brackets mark lncRNA and TE-linked long transcripts domains. See also Figure S3.

Figure 4. PAF1 antagonizes PIWI silencing

(A) ChIP/qPCR analyses of H3K4me3 and H3K9me3 on OSS cell loci and reporters after siRNA knockdown and reporter transfection. Left set of columns represent cells transfected with Sense Flam reporters, right set are cells with the Antisense Flam reporters. Standard deviation of triplicate measurements from biological duplicates, with significant differences compared to siGFP control; *=p<0.05, t-test. (B) Assay results for Flam reporter (top) and a mdg1-3'end segment reporter (bottom). Standard deviation of biological triplicates with significant differences to the siGFP control; *=p<0.05, **=p<0.01, t-test. Additional bars mark tests between siPIWI and siPAF1 to siPIWI+siPAF1. (C) A model proposing that direct factors assist PIWI in targeting nascent and maturing transcripts, with silencing capacity influenced by proximity of the piRNA-targeted element to the promoter. PAF1/RTF1 reduction may stall nascent RNA release to enhance PIWI silencing. See also Figure S4.