Arginine methylation of DDX5 RGG/RG motif by PRMT5 regulates RNA:DNA resolution

Sy, Mersaoui; Z, Yu; Coulombe, Yan; Karam, Marc; Ff, Busatto; Masson, Jean‐Yves; Richard, Stéphane

doi:10.1101/451823

Cited by 41 publications

(98 citation statements)

References 66 publications

(98 reference statements)

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“…In human cells, mtSSB has been reported to localize partially to RNA granules (64), whilst defects in the machinery of RNA processing and degradation results in the accumulation of persistent R-loops, resulting in RNase H1 recruitment to nucleoids (65). In an earlier study, vertebrate RNase H1 was not observed as a nucleoid protein (66), consistent with its interactions with mitochondrial replication and RNA processing enzymes being transient, and mediated by its association with RNA/DNA hybrid substrate, rather than by direct protein-protein interactions Amongst other nuclear hits, we identified a second component of the R-loop processing machinery, Rm62, the Drosophila homologue of human DDX5 (67). This suggests a potential interaction between two independent machineries to resolve R-loops.…”

Section: Significance Of Rnase H1 Interactorssupporting

confidence: 73%

A second hybrid-binding domain modulates the activity of Drosophila ribonuclease H1

Cózar

Carretero-Junquera

Ciesielski

et al. 2020

Preprint

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In eukaryotes, ribonuclease H1 (RNase H1) is involved in the processing and removal of RNA/DNA hybrids in both nuclear and mitochondrial DNA. The enzyme comprises a C-terminal catalytic domain and an N-terminal hybrid-binding domain (HBD), separated by a linker of variable length, which in Drosophila melanogaster (Dm) is exceptionally long, 115 amino acids. Molecular modeling predicted this extended linker to fold into a structure similar to the conserved HBD. We measured catalytic activity and substrate binding by EMSA and biolayer interferometry, using a deletion series of protein variants. Both the catalytic domain and the conserved HBD were required for high-affinity binding to heteroduplex substrates, whilst loss of the novel HBD led to a ~90% drop in K[cat] with a decreased K[M], and a large increase in the stability of the RNA/DNA hybrid-enzyme complex. The findings support a bipartite binding model for the enzyme, whereby the second HBD facilitates dissociation of the active site from the product, allowing for processivity. We used shotgun proteomics to identify protein partners of the enzyme involved in mediating these effects. Single-stranded DNA-binding proteins from both the nuclear and mitochondrial compartments, respectively RpA-70 and mtSSB, were prominently detected by this method. However, we were not able to document direct interactions between mtSSB and Dm RNase H1 when co-overexpressed in S2 cells, or functional interactions in vitro. Further studies are needed to determine the exact reaction mechanism of Dm RNase H1, the nature of its interaction with mtSSB and the role of the second HBD in both.

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Section: Significance Of Rnase H1 Interactorssupporting

confidence: 73%

A second hybrid-binding domain modulates the activity of Drosophila ribonuclease H1

Cózar

Carretero-Junquera

Ciesielski

et al. 2020

Preprint

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“…The helicase Rm62 interacts genetically with both PcG and TrxG genes, and colocalizes with the PRE-binding protein Dsp1 on polytene chromosomes 38 . Rm62 is the Drosophila homologue of the DDX5 helicase, which can unwind RNA-DNA hybrids in vitro and is implicated in R-loop resolution in vivo 39 . A recent genome-wide RNAi screen for TrxG interacting genes (which should antagonize PcG function) identified the gene for RNaseH1 40 .…”

Section: Discussionmentioning

confidence: 99%

RNA-DNA strand exchange by the Drosophila Polycomb complex PRC2

et al. 2020

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Polycomb Group (PcG) proteins form memory of transient transcriptional repression that is necessary for development. In Drosophila, DNA elements termed Polycomb Response Elements (PREs) recruit PcG proteins. How PcG activities are targeted to PREs to maintain repressed states only in appropriate developmental contexts has been difficult to elucidate. PcG complexes modify chromatin, but also interact with both RNA and DNA, and RNA is implicated in PcG targeting and function. Here we show that R-loops form at many PREs in Drosophila embryos, and correlate with repressive states. In vitro, both PRC1 and PRC2 can recognize R-loops and open DNA bubbles. Unexpectedly, we find that PRC2 drives formation of RNA-DNA hybrids, the key component of R-loops, from RNA and dsDNA. Our results identify R-loop formation as a feature of Drosophila PREs that can be recognized by PcG complexes, and RNA-DNA strand exchange as a PRC2 activity that could contribute to R-loop formation.

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“…While this work used normally cultured, unperturbed human cells, many studies have used S9.6-based imaging to measure changes in RNA:DNA hybrid content under various experimental conditions. An array of gene perturbations and drug treatments have been reported to cause changes in S9.6 IF signal attributed to altered R-loop metabolism or RNA:DNA hybrid levels [8,30,[34][35][36][47][48][49][50]. Many of the genes that have been ascribed roles in R-loop regulation participate in diverse RNA metabolic processes like transcription, RNA splicing, RNA processing, and RNA export [6].…”

Section: Discussionmentioning

confidence: 99%

Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids

Chédin

Smolka

Sanz

et al. 2020

Preprint

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The contribution of RNA:DNA hybrid metabolism to cellular processes and disease states has become a prominent topic of study. The S9.6 antibody recognizes RNA:DNA hybrids with a subnanomolar affinity, making it a broadly used tool to detect and study RNA:DNA hybrids. However, S9.6 also binds double-stranded RNA in vitro with significant affinity. Though frequently used in immunofluorescence microscopy, the possible reactivity of S9.6 with non-RNA:DNA hybrid substrates in situ, particularly RNA, has not been comprehensively addressed. Furthermore, S9.6 immunofluorescence microscopy has been methodologically variable and generated discordant imaging datasets. In this study, we find that the majority of the S9.6 immunofluorescence signal observed in fixed human cells arises from RNA, not RNA:DNA hybrids. S9.6 staining was quantitatively unchanged by pre-treatment with the human RNA:DNA hybrid-specific nuclease, RNase H1, despite experimental verification in situ that S9.6 could recognize RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to pre-treatments with RNase T1, and in some cases RNase III, two ribonucleases that specifically degrade singlestranded and double-stranded RNA, respectively. In contrast, genome-wide maps obtained by high-throughput DNA sequencing after S9.6-mediated DNA:RNA Immunoprecipitation (DRIP) are RNase H1-sensitive and RNase T1-and RNase IIIinsensitive. Altogether, these data demonstrate that the S9.6 antibody, though capable of recognizing RNA:DNA hybrids in situ and in vitro, suffers from a lack of specificity that precludes reliable imaging of RNA:DNA hybrids and renders associated imaging data inconclusive in the absence of controls for its promiscuous recognition of cellular RNAs.

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Arginine methylation of DDX5 RGG/RG motif by PRMT5 regulates RNA:DNA resolution

Cited by 41 publications

References 66 publications

A second hybrid-binding domain modulates the activity of Drosophila ribonuclease H1

A second hybrid-binding domain modulates the activity of Drosophila ribonuclease H1

RNA-DNA strand exchange by the Drosophila Polycomb complex PRC2

Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids

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