DICER ribonuclease removes harmful R-loops

Abstract

R-loops, which consist of a DNA-RNA hybrid and a displaced DNA strand, are known to threaten genome integrity. To counteract this, different mechanisms suppress R-loop accumulation by either preventing the hybridization of RNA with the DNA template (RNA biogenesis factors), unwinding the hybrid (DNA-RNA helicases), or degrading the RNA moiety of the R-loop (type H ribonucleases [RNases H]). Thus far, RNases H are the only nucleases known to cleave DNA-RNA hybrids. Now, we show that the RNase DICER also resolves R-loops. Biochemical analysis reveals that DICER acts by specifically cleaving the RNA within R-loops. Importantly, a DICER RNase mutant impaired in R-loop processing causes a strong accumulation of R-loops in cells. Our results thus not only reveal a function of DICER as an R-loop resolvase independent of DROSHA but also provide evidence for the role of multi-functional RNA processing factors in the maintenance of genome integrity in higher eukaryotes.

Keywords: DICER; DICER1 syndrome; DNA damage; R-loops; RNA nuclease; genetic instability; replication fork stalling.

Figure 1.. Screening for RNA related factors involved in R-loop homeostasis

(A) Relative S9.6 signal levels detected by immunofluorescence in 96-well plates in HeLa cells after the indicated siRNA treatments. The signal analysis was performed with Imagexpress micro high content screening system (Molecular Devices). (B) S9.6 signal detection relative levels after the indicated siRNA treatments in cells transfected with either empty pEGFP-C1 (−) or pEGFP-M27 (+), which expresses RNase H1 (RH1). Relative nuclear S9.6 signal intensity after nucleolus signal removal in 150–2000 total cells from three independent experiments are shown. The median of each population is shown. ***p<0.001 (Mann-Whitney U test). (C) Comet tail moment from single-cell alkaline gel electrophoresis (comet assay) of HeLa cells transfected with the indicated siRNAs and either empty pEGFP-C1 (−) or pEGFPM27 (+), which expresses RNase H1 (RH1). More than 220 total cells were analyzed. The median of each population and representative images are shown *p<0.05, **p<0.01, ***p<0.001 (Mann-Whitney U test).

Figure 2.. DICER depletion specifically leads to dsRNA and DNA-RNA hybrid accumulation in the nucleus.

(A) Nuclear S9.6 signal detection levels after the indicated siRNA treatments. The data correspond to more than 400 total cells from 3 independent experiments. B) Quantification of protein and RNA levels as determined by western blot (top) and RT-PCRs (bottom) after the indicated siRNA treatments (C) J2 signal detection levels after the indicated siRNA treatments plus in vitro treatment with RNase III performed preimmunofluorescence (RIII) where indicated. The data correspond to 200 total cells from two independent experiments. (D) S9.6 signal detection levels after the indicated siRNA treatments transfected with either empty pEGFP-C1 (−) or pEGFP-M27 (+), which expresses RNase H1 (RH1). All samples were treated pre-immunofluorescence in vitro with RNase III (RIII). The data correspond to more than 260 total cells from two independent experiments. (E) S9.6 signal detection levels after the indicated siRNA treatments and in vitro treatments performed pre-immunofluorescence with RNase III (RIII) and/or RNase H (RH) as indicated. The data correspond to more than 300 total cells from two independent experiments. In all panels, boxes and whiskers indicate 10–90 percentiles. Representative images are shown in which single color channel levels were adjusted to the same extent in the entire image to allow visualization. **p<0.01 ***p<0.001, ****p<0.0001 (Mann-Whitney U test).

Figure 3. DNA-RNA hybrid immunoprecipitation in DICER-depleted cells.

(A) Relative DRIP-qPCR signal values at APOE, RPL13A, HBE1, HMGA1, MALAT1, RPPH1, and 18S rDNA genes in HeLa cells transfected with siC or siDICER and treated pre-immunoprecipitation in vitro with RNase H (RH) where indicated. The mean ± SEM from 3–7 independent experiments is shown. (B) Relative DRIP-qPCR signal values at APOE and RPL13A genes in HeLa cells transfected with siC or siDICER, treated pre-immunoprecipitation in vitro with RNase III (RIII) and with RNase H (RH) where indicated. The data correspond to 3–4 independent experiments. (C) Relative DRIP-qPCR signal values at RPL13A gene in K562 cells transfected with siC or siDICER, treated pre-immunoprecipitation in vitro with RNase III (RIII) and with RNase H (RH) where indicated. The data correspond to 11 independent experiments. In all panels *p<0.05, **p<0.01, ***p<0.001 (one-tailed paired T-test). On top of each histogram, diagrams of the DNA regions include the sites for the restriction enzymes that were used in the experiment (BsrGI, EcoRI, HindIII, SspI, XbaI) as dashed vertical lines as well as the position of the PCR products used for detection (red horizontal lines). The scale of the region can be appreciated by the size of the grey horizontal lines, which correspond to 500 bp.

Figure 4.. DICER RNase activity on 30bp, 25 bp, and 35 bp R-loops.

(A) Scheme of DICER domains and mutations relevant in this study. (B) Purification of DICER-WT and DICER mutants R3Am (D1320A, E1564A), R3Bm (D1709A) and R3ABm (D1320A, E1564A, D1709A) (C) Native gel showing RNase activity of DICER-WT and DICER mutants R3Am (D1320A, E1564A), R3Bm (D1709A) and R3ABm (D1320A, E1564A, D1709A) on 30bp, 25 bp, and 35 bp R-loops with no-RNA overhang (D) Denaturing gel showing RNase activity of DICER-WT and DICER mutants R3Am (D1320A, E1564A), R3Bm (D1709A) and R3ABm (D1320A, E1564A, D1709A) on 30bp, 25 bp, and 35 bp R-loops with no-RNA overhang.

Figure 5.. Specificity of R-loop binding and cleavage by DICER.

(A) R-loop binding by DICER-WT and DICER-R3ABm. (B) Binding of R-loop, D-loop and DNA-RNA with flanking dsDNA by DICER-WT. (C) Binding of DNA bubble and ssDNA by DICER-WT. (D) RNase activity of DICER-WT on R-loops, D-loops and DNA-RNA hybrids with flanking dsDNA. In all panels, the quantification of 3 independent experiments is shown.

Figure 6.. DICER action on DNA-RNA hybrids in cells depends on its RNase activity.

S9.6 signal detection levels in HeLa cells transfected with siC, or siDICER and with the empty plasmid pcDNA3(−) or either pDICERr or pDICERr-R3ABm, which overexpress siDICER.1-resistant versions of DICER WT or R3ABm mutant. All samples were treated in vitro pre-immunofluorescence with RNase III (RIII) and/or RNase H (RH) as indicated. The data correspond to 700 total cells from 5 independent experiments. Representative images are shown in which single color channel levels were adjusted to the same extent in the entire image to allow visualization.

Figure 7.. DICER overexpression suppresses R-loop accumulation in cells.

(A) S9.6 signal detection levels in HeLa cells transfected with siC or siTHOC1 and with either empty pcDNA3(−) or pDICER (+), which overexpresses DICER. All samples were treated in vitro with RNase III (RIII) pre-immunofluorescence. The data correspond to 600 total cells from 9 independent experiments. Representative images are shown. (B) Replication fork asymmetry as measured by DNA combing assay in HeLa cells transfected with siC or siTHOC1 and with either the empty plasmid pcDNA3(−) or pDICER (+), which overexpresses DICER. The data correspond to more than 34 measurements in total in each condition from two independent experiments. The median of each population is indicated. A scheme and representative fibers from each condition are shown on top. (C) S9.6 signal levels in HeLa cells transfected with siC, siFANCD2 or siUAP56 and with the empty plasmid pcDNA3(−) or pDICER (+), which overexpresses DICER. All samples were treated in vitro with RNase III (RIII) pre-immunofluorescence. The data correspond to 500–1400 total cells from 7 independent experiments. Representative images are shown in which single color channel levels were adjusted to the same extent in the entire image to allow visualization. In A and C, boxes and whiskers indicate 10–90 percentiles. ***p<0.001, ****p<0.0001 (Mann-Whitney U test). (D) Model integrating the different molecular activities of DICER in D-loops, R-loops or DNA-RNA hybrids, in which it is shown that DICER is able to bind D-loops and R-loops but not DNA-RNA hybrids and cleaves R-loops but not D-loops.