The molecular chaperone ALYREF promotes R-loop resolution and maintains genome stability

Abstract

Unscheduled R-loops usually cause DNA damage and replication stress, and are therefore a major threat to genome stability. Several RNA processing factors, including the conserved THO complex and its associated RNA and DNA-RNA helicase UAP56, prevent R-loop accumulation in cells. Here, we investigate the function of ALYREF, an RNA export adapter associated with UAP56 and the THO complex, in R-loop regulation. We demonstrate that purified ALYREF promotes UAP56-mediated R-loop dissociation in vitro, and this stimulation is dependent on its interaction with UAP56 and R-loops. Importantly, we show that ALYREF binds DNA-RNA hybrids and R-loops. Consistently, ALYREF depletion causes R-loop accumulation and R-loop-mediated genome instability in cells. We propose that ALYREF, apart from its known role in RNA metabolism and export, is a key cellular R-loop coregulator, which binds R-loops and stimulates UAP56-driven resolution of unscheduled R-loops during transcription.

Keywords: ALYREF; DNA-RNA helicase; R-loop; TREX complex; UAP56; genome instability.

Figure 1

ALYREF promotes the R-loop dissociation activity of UAP56.A, the R-loop dissociation activity was characterized with a serial dilution of UAP56 protein using DNA-RNA flap structures mimicking R loops as substrates. The positions of the R-loop substrates and unwound products (duplex DNA) are indicated at the right, where the stars show the position of the radiolabel. Gels were dried and subject to phosphorimaging analysis. The bottom graph shows the percentage of dsDNA product recovered after the reaction respect to the UAP56 concentration-dependent manner. B, ALYREF significantly promotes the R-loop dissociation activity of UAP56. ALYREF (6 or 12 nM) was incubated with UAP56 (25 or 50 nM, lanes 4–7) and used in the R-loop dissociation assay. ALYREF alone (lanes 8–9) or UAP56 alone (lanes 2–3) were tested for R-loop dissociation as control. Other details are in A. The percentage of dsDNA product recovered after the reaction were quantified as the mean values ± SD (n = 3 technical replicates). ∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001 (two-tailed paired t test).

Figure 2

ALYREF binds to DNA-RNA hybrids and R-loops in vitro. Increasing amounts of ALYREF was incubated with 5 nM of ssRNA (A), ssDNA (B), dsRNA (C), dsDNA (D), DNA-RNA hybrids (E), R-Flap (F) or R-loop (G) structures, and the reaction mixtures were resolved in 7% polyacrylamide gels at 4 °C, and pictures of representative gels are shown. H, the percentage of the bound substrates was calculated as the percentage of intensity reduction of free DNA (or RNA) structure and was shown as the mean values ± SD (n = 3 technical replicates). Two-tailed paired t test between the percentage of R-loop and dsDNA binding was performed. ∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001.

Figure 3

Isolation of ALYREF mutants impaired for UAP56 interaction or nucleic acid binding.A, domain organization of ALYREF. The N- and C-terminal UAP56-binding motifs (UBMs), the two RGG motifs (RGG), and the RNA recognition motif (RRM) are indicated for ALYREF. The constructs of ALYREF-ΔNC, which depletes both the N- and C-terminal UBMs, and ALYREF-10E, which mutates ten conserved arginine (R) to glutamic acids (E), are shown. B, SDS-PAGE analysis of purified ALYREF and its mutants ALYREF-ΔNC and ALYREF-10E. C, Ni-NTA pull-down assay showing that ALYREF-ΔNC is defective in interaction with UAP56, whereas ALYREF-10E is only slightly affected in this regard. Purified His₆-tagged ALYREF, ALYREF-ΔNC, or ALYREF-10E proteins bound to Ni-NTA beads were examined for their abilities to pull down UAP56. Proteins were examined by SDS-PAGE, and pictures of representative gels after Coomassie blue stain are shown. The relative ratio of bound UAP56 in the eluate (E) is indicated. D and E, electrophoretic mobility shift assay showing that ALYREF-ΔNC was proficient to bind R-loop structure (D), but ALYREF-10Ε was defective for R-loop binding (E). Increased amounts of WT ALYREF or ALYREF mutants (ALYREF-ΔNC or ALYREF-10Ε) were incubated with DNA-RNA flap structure mimicking R loops (5 nM) for protein–R-loop complex formation. The percentage of bound R-loops was quantified as the mean values ± SD (n = 3 technical replicates). ∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001 (two-tailed paired t test). Black stars denote significant increases, whereas red stars denote significant decreases. S, supernatant; UBM, UAP56-binding motif; W, wash.

Figure 4

The promotion of UAP56-mediated R-loop resolution by ALYREF requires its interaction with UAP56.A, ALYREF, but not its UAP56 interaction defective mutant ALYREF-ΔNC, was able to stimulate the UAP56-mediated R-loop dissociation. ALYREF or ALYREF-ΔNC (3 or 6 nM) were incubated with UAP56 (60 nM, lanes 5–6, 9–10) and used in the R-loop dissociation assay. UAP56 alone (60 nM, lane 2), ALYREF alone (3–6 nM, lanes 3–4), or ALYREF-ΔNC alone (3–6 nM, lanes 7–8) were tested for R-loop dissociation as control. The percentage of dsDNA product recovered after the reaction were quantified as the mean values ± SD (n = 3 technical replicates). ∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001 (two-tailed paired t test). B and C, a time course of R-loop dissociation assay showing that ALYREF, but not ALYREF-ΔNC, stimulated the UAP56-mediated R-loop dissociation. The R-loop dissociation were compared by UAP56 (60 nM) alone, ALYREF (6 nM) or ALYREF-ΔNC (6 nM) alone, UAP56 (60 nM) and ALYREF (6 nM) together, and UAP56 (60 nM) and ALYREF-ΔNC (6 nM) together, at 5, 10, and 15 min. The pictures of representative gels are shown. D, graphical representation shows the mean of the percentage of dsDNA product recovered after the reaction ± SD from at least three independent experiments from B and C (n = 3 technical replicates). ∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001 (two-tailed paired t test).

Figure 5

The promotion of UAP56-mediated R-loop resolution by ALYREF requires its interaction with R-loops and nucleic acids.A, ALYREF, but not its nucleic acids interaction defective mutant ALYREF-10E, was able to stimulate the UAP56-mediated R-loop dissociation. ALYREF or ALYREF-10E (3 or 6 nM) were incubated with UAP56 (60 nM, lanes 5–6, 9–10) and used in the R-loop dissociation assay. UAP56 alone (60 nM, lane 2), ALYREF alone (3–6 nM, lanes 3–4), or ALYREF-10E alone (3–6 nM, lanes 7–8) were tested for R-loop dissociation as control. The percentage of dsDNA product recovered after the reaction were quantified as the mean values ± SD (n = 3 technical replicates). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 (two-tailed paired t test). B and C, a time course of R-loop dissociation assay showing that ALYREF, but not ALYREF-10E, stimulated the UAP56-mediated R-loop dissociation. The R-loop dissociation were compared by UAP56 (60 nM) alone, ALYREF (6 nM) or ALYREF-10E (6 nM) alone, UAP56 (60 nM) and ALYREF (6 nM) together, and UAP56 (60 nM) and ALYREF-10E (6 nM) together, at 5, 10, and 15 min. The pictures of representative gels are shown. D, graphical representation shows the mean of the percentage of dsDNA product recovered after the reaction ± SD from at least three independent experiments from B and C (n = 3 technical replicates). ∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001 (two-tailed paired t test).

Figure 6

ALYREF prevents R-loop accumulation and R-loop–mediated genome instability.A, representative images of immunostaining with S9.6 (red) and anti-nucleolin (green) antibodies in U2OS cells upon ALYREF knockdown. The median of S9.6 signal intensity per nucleus after nucleolar signal removal in siRNAs U2OS transfected cells and in vitro treated with RNase III and with or without RNase H is shown. Data from more than 200 total cells from three independent experiments is shown. ∗∗∗∗p < 0.0001 (Mann–Whitney U test, two-tailed). Black stars denote significant increases, whereas red stars denote significant decreases. B, relative DRIP-qPCR signal values in siALY U2OS cells at the indicated regions is shown. Samples were treated in vitro with RNase H prior immunoprecipitation where indicated. Signal values were normalized with respect to the siC control cells in each experiment and the mean ± SEM is plotted (n = 5). ∗p < 0.05 and ∗∗p < 0.01 (one-tailed paired t test). Schematic diagrams of APOE, RPL13A, and MIB2 genes are shown. Exons are depicted as closed boxes, the arrows indicate the start of transcription, and in red are indicated the regions where the primer pairs used for amplification were located. C, representative images of single-cell alkaline gel electrophoresis (comet assay) of U2OS cells upon ALYREF depletion with and without RNase H1 overexpression. Values were normalized with respect to the siC control cells in each experiment. Data are plotted as mean of the medians ± SEM (n = 6). More than 80 cells per condition were analyzed in each experiment. ∗p < 0.05 (one-tailed paired t test). D, model to explain the role of ALYREF in R-loop resolution during transcription. When ALYREF is present in normal amount in cells, it would bind to the R-loop structures through its two RGG domains, stimulating the interaction of UAP56 to cotranscriptionally formed R-loops through the two N- and C- terminal UBMs of ALYREF. UAP56 could be orientated or oligomerized by ALYREF in an active conformation for R-loop dissociation, which ensures that unscheduled R-loops are resolved cotranscriptionally to maintain genome stability (left). Instead, in siALY cells, UAP56 could not efficiently dissociate unscheduled R-loops without ALYREF, causing R-loop accumulation and R-loop–mediated genome instability (right). DRIP, DNA-RNA immunoprecipitation; qPCR, quantitative PCR; UBM, UAP56-binding motif.