Dynamic nucleoplasmic and nucleolar localization of mammalian RNase H1 in response to RNAP I transcriptional R-loops

Shen, Wen‐Hui; Sun, Hong; Hoyos, Cheryl Li De; Bailey, Jeffrey K; Liang, Xiaojing; Crooke, Stanley T.

doi:10.1093/nar/gkx710

Cited by 51 publications

(76 citation statements)

References 75 publications

(103 reference statements)

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“…Nuclear staining was predominantly nucleolar in morphology and localization. These staining patterns are congruent with several previous reports [26,28,30,41] and have been explained by claims that ribosomal DNA is a hotspot for R-loop formation in the nucleus [42] and that mitochondrial genomes in the cytoplasm contain R-loops [43]. To determine if the cytoplasmic S9.6 signal derives from mitochondria, we labeled mitochondria prior to fixation using the vital dye, MitoTracker…”

Section: Rna Constitutes the Majority Of The S96 Immunofluorescence supporting

confidence: 88%

“…This has made the use of ribonuclease H (RNase H) enzymes, nucleases that specifically degrade the RNA strand of RNA:DNA hybrids, critical in verifying the RNA:DNA hybrid-dependence of measurements made using S9.6-based assays [22]. While RNase H pre-treatments are routinely used as negative controls in molecular S9.6-based methods like immunoprecipitations and dot blots [23], cellular imaging using S9.6 is frequently performed with no enzymatic controls [8,[24][25][26][27][28]. When RNase H controls are implemented, results vary from study to study, with some studies reporting removal of S9.6 immunofluorescence signal by exogenous RNase H treatment and others finding RNase H-resistant signal [29][30][31][32][33][34].…”

Section: Introductionmentioning

confidence: 99%

“…Lastly, though a common goal of using S9.6 is to image R-loop structures in the nucleus, cytoplasmic S9.6 signal has been consistently observed across studies (see references above). This signal is often unaddressed or attributed to R-loops arising from the mitochondrial genome [13,28].…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Rna Constitutes the Majority Of The S96 Immunofluorescence supporting

confidence: 88%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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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“…Mammalian cells have two enzymes with the ability to degrade the RNA strand of RNA:DNA heteroduplexes: RNase H1 and RNase H2 (reviewed by Cerritelli and Crouch, 2009). RNase H1 has two isoforms: one localises to mitochondria and is essential for mitochondrial DNA replication (Cerritelli et al, 2003), and the second is nuclear and important for preventing R-loops and consequent transcription-replication conflicts (Nguyen et al, 2017;Parajuli et al, 2017;Shen et al, 2017). RNase H2, a heterotrimeric complex, is the predominant nuclear enzyme responsible for RNA:DNA hybrid degradation (reviewed by Reijns and Jackson, 2014), although this may depend on cell type.…”

Section: Introductionmentioning

confidence: 99%

RNase H2, mutated in Aicardi‐Goutières syndrome, promotes LINE‐1 retrotransposition

et al. 2018

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Long INterspersed Element class 1 (LINE‐1) elements are a type of abundant retrotransposons active in mammalian genomes. An average human genome contains ~100 retrotransposition‐competent LINE‐1s, whose activity is influenced by the combined action of cellular repressors and activators. TREX1, SAMHD1 and ADAR1 are known LINE‐1 repressors and when mutated cause the autoinflammatory disorder Aicardi‐Goutières syndrome (AGS). Mutations in RNase H2 are the most common cause of AGS, and its activity was proposed to similarly control LINE‐1 retrotransposition. It has therefore been suggested that increased LINE‐1 activity may be the cause of aberrant innate immune activation in AGS. Here, we establish that, contrary to expectations, RNase H2 is required for efficient LINE‐1 retrotransposition. As RNase H1 overexpression partially rescues the defect in RNase H2 null cells, we propose a model in which RNase H2 degrades the LINE‐1 RNA after reverse transcription, allowing retrotransposition to be completed. This also explains how LINE‐1 elements can retrotranspose efficiently without their own RNase H activity. Our findings appear to be at odds with LINE‐1‐derived nucleic acids driving autoinflammation in AGS.

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“…In mitochondria, RNase H1 has been implicated in mtDNA replication initiation and segregation (2,26,27), as well in mitochondrial RNA processing (2,28,29) and RNA/DNA removal (30). In the nucleus, it has been proposed to be essential for some types of DNA repair (31, 32), removal of persistent heteroduplex (33,34) and telomere maintenance (35).…”

Section: Introductionmentioning

confidence: 99%

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.

show abstract

Dynamic nucleoplasmic and nucleolar localization of mammalian RNase H1 in response to RNAP I transcriptional R-loops

Cited by 51 publications

References 75 publications

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

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

RNase H2, mutated in Aicardi‐Goutières syndrome, promotes LINE‐1 retrotransposition

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

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