Human mitochondrial degradosome prevents harmful mitochondrial R loops and mitochondrial genome instability

Abstract

R loops are nucleic acid structures comprising an DNA–RNA hybrid and a displaced single-stranded DNA. These structures may occur transiently during transcription, playing essential biological functions. However, persistent R loops may become pathological as they are important drivers of genome instability and have been associated with human diseases. The mitochondrial degradosome is a functionally conserved complex from bacteria to human mitochondria. It is composed of the ATP-dependent RNA and DNA helicase SUV3 and the PNPase ribonuclease, playing a central role in mitochondrial RNA surveillance and degradation. Here we describe a new role for the mitochondrial degradosome in preventing the accumulation of pathological R loops in the mitochondrial DNA, in addition to preventing dsRNA accumulation. Our data indicate that, similar to the molecular mechanisms acting in the nucleus, RNA surveillance mechanisms in the mitochondria are crucial to maintain its genome integrity by counteracting pathological R-loop accumulation.

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

“…Cytoplasmic S9.6 staining had a distribution more similar to HSP27, which labels the entire cell body, than to mitochondria ( Figure 1B), and S9.6 immunofluorescence signal did not show enrichment in or correlation with mitochondrial territories ( Figure 1C). These observations agree with prior work in human cells grown under standard conditions [32], and indicate that mitochondria are not the source of the majority of cytoplasmic S9.6 staining, and that S9.6 detects extra-mitochondrial structures throughout the cytoplasm.…”

“…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]. Additionally, the S9.6 staining pattern itself varies from study to study, often coincident with methodological differences in fixation, permeabilization, and buffers used to prepare and/or enzymatically treat cells prior to immunolabeling [8,24,29,30,32,35,36].…”

“…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]. Additionally, the S9.6 staining pattern itself varies from study to study, often coincident with methodological differences in fixation, permeabilization, and buffers used to prepare and/or enzymatically treat cells prior to immunolabeling [8,24,29,30,32,35,36]. 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).…”

Arginine methylation of the DDX 5 helicase RGG / RG motif by PRMT 5 regulates resolution of RNA:DNA hybrids

“…Physiologically, R-loops or RNA:DNA hybrids are programmed structures that occur during many cellular processes, including transcription, replication, and immunoglobulin class switching (Skourti-Stathaki & Proudfoot, 2014;Chedin, 2016;Bhatia et al, 2017). Persistent R-loops impede DNA replication and if unresolved ultimately cause DNA breaks and genomic instability (Hamperl & Cimprich, 2016;Aguilera & Gomez-Gonzalez, 2017) or mitochondrial instability (Silva et al, 2018). Therefore, it is not surprising that there are specialized machineries or protein complexes to prevent and resolve these R-loops.…”

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

“…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).…”