Small molecule inhibition of CPSF3 impacts R-loop distribution and abundance

Abstract

R-loops are three-stranded nucleic acid structures consisting of an RNA/DNA hybrid and a displaced strand of DNA. These structures have been implicated in a variety of regulatory cellular processes. Their untimed or excess accumulation, however, can cause genomic instability and induce DNA damage. Most R-loops form co-transcriptionally when the nascent transcript reanneals to unwound DNA duplex. Changes in the rate of transcription have the potential to impact R-loop formation, and compounds that modulate R-loop formation would be useful molecular tools and therapeutic leads. Cleavage and Polyadenylation Specific Factor 3 (CPSF3) recognizes the pre-mRNA 3' cleavage site, cleaves the transcript prior to polyadenylation, and has been linked to R-loop formation. Inhibition of CPSF3 has been found to induce transcriptional readthrough and cell proliferation defects. A previous report suggested that inhibition of CPSF3 with a small molecule causes a global increase in R-loop abundance. Here we test the impact of YT-II-100, a novel inhibitor of CPSF3. We find that addition of YT-II-100 increases global R-loop formation but does not change R-loop formation at specific genes that are normally used as positive controls for R-loop formation. We performed parallel assays using previously reported compound JTE-607 and observed similar results. Our data emphasize the need for cautious interpretation of experiments using JTE-607 and YT-II-100. There may be different mechanisms of R-loop formation depending on gene loci, with the control of R-loop formation at some genes diverging from the regulation of global R-loop formation.

Figure 1.

Inhibition of CPSF3 with YT-II-100 leads to a global increase in R-loops A) Chemical structure of YT-II-100. B) Fluorescence microscopy images of wild-type cells staining for RNA/DNA hybrids (green) and DAPI/cell nuclei (blue) following 6 hours of DMSO or YT-II-100 treatment. C) Dot blot staining for RNA/DNA hybrids of wild-type and Y207H HCT116 cells treated with DMSO or YT-II-100 for 6 hours +/− RNase H treatment (N=3).

Figure 2.

Inhibition of CPSF3 with JTE-607 leads to a global increase in R-loops A) Chemical structure of JTE-607 B) Fluorescence microscopy images of wild-type cells staining for RNA/DNA hybrid (green) and DAPI/cell nuclei (blue) following 6 hours of DMSO or JTE-607 treatment. C) Dot blot probing for RNA/DNA hybrids of wild-type and Y207H HCT116 cells treated with DMSO or JTE-607 for 6 hours +/− RNase H treatment (N=3).

Figure 3.

Inhibition of CPSF3 with YT-II-100 has mixed influence on specific R-loop loci. DRIP-qPCR data plotted as fold enrichment relative to negative control gene (SNRPN) of (A) WT and (B) Y207H cells treated with DMSO or YT-II-100 for 6 hours. Samples that were pre-treated with RNase H are denoted as RH. Points are plotted as the average of biological replicates +/− SD. Significance was analyzed by Mann-Whitney test and denoted with *: EGR1 P=0.0286

Figure 4.

Inhibition of CPSF3 with JTE-607 has mixed influence on specific R-loop loci. DRIP-qPCR data plotted as fold enrichment relative to negative control gene (SNRPN) of (A) WT and (B) Y207H cells treated with DMSO or JTE-607 for 6 hours. Samples that were pre-treated with RNase H are denoted as RH. Points are plotted as the average of biological replicates +/− SD. Significance was analyzed by Mann-Whitney test.