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Human Mitochondrial Degradosome Prevents Harmful Mitochondrial R Loops and Mitochondrial Genome Instability

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Human Mitochondrial Degradosome Prevents Harmful Mitochondrial R Loops and Mitochondrial Genome Instability Human mitochondrial degradosome prevents harmful mitochondrial R loops and mitochondrial genome instability Sonia Silvaa, Lola P. Caminoa, and Andrés Aguileraa,1 aCentro Andaluz de Biología Molecular y Medicina Regenerativa, Universidad de Sevilla–Consejo Superior de Investigaciones Científicas–Universidad Pablo de Olavide, 41092 Seville, Spain Edited by Uttiya Basu, Columbia University, and accepted by Editorial Board Member Douglas Koshland September 13, 2018 (received for review April 26, 2018) R loops are nucleic acid structures comprising an DNA–RNA hybrid and reduce its ability to hybridize with the negatively supercoiled and a displaced single-stranded DNA. These structures may occur DNA template behind the traveling RNA polymerase (13, 14). transiently during transcription, playing essential biological func- Indeed, other factors involved in the RNA packing into messenger tions. However, persistent R loops may become pathological as ribonucleoprotein complexes (mRNPs), splicing and export of the they are important drivers of genome instability and have been asso- mature mRNA can lead to the accumulation of harmful R loops, ciated with human diseases. The mitochondrial degradosome is a func- indicating that RNA biogenesis and export in general contribute tionally conserved complex from bacteria to human mitochondria. It is to prevent R-loop accumulation (15, 16). Additionally, cells have composed of the ATP-dependent RNA and DNA helicase SUV3 and the also developed means to deal with R loops once they form. These PNPase ribonuclease, playing a central role in mitochondrial RNA sur- include a growing number of RNA and DNA helicases, such as veillance and degradation. Here we describe a new role for the Senataxin and DDX23, which unwind the RNA hybridized from mitochondrial degradosome in preventing the accumulation of the DNA molecule, with yet unknown genomic locus specificity, if pathological R loops in the mitochondrial DNA, in addition to prevent- any (5, 17). The best-studied R-loop–removing factors, and likely ing dsRNA accumulation. Our data indicate that, similar to the molec- most important, are the RNase H endonucleases, which are pre- ular mechanisms acting in the nucleus, RNA surveillance mechanisms sent in most organisms and specifically target and degrade the in the mitochondria are crucial to maintain its genome integrity RNA moiety within the DNA–RNA hybrids (18). RNase H1, in by counteracting pathological R-loop accumulation. particular, has been proven central for proper DNA replication in the nucleus, removing Okazaki fragments, and in the mitochon- genome instability | mitochondrial degradosome | R loops | dria, where replication initiation relies on a persistent R loop used RNA metabolism | transcription–replication conflicts as a primer for the replicative DNA polymerase (19). In addition, well-established genome guardians from the DNA damage re- ery-short DNA–RNA hybrids occur during both replication sponse and repair pathways have also been implicated in reducing Vand transcription, serving specific purposes in DNA or RNA harmful R loops associated with replication fork stalling, such as metabolism, such as priming lagging-strand DNA synthesis or BRCA1, BRCA2, and Fanconi anemia factors, which likely deal extending the nascent RNA inside the active pocket of RNA with persistent and unresolved R loops in cells entering the S–G2 polymerases. R loops, on the other hand, are a distinct class of phase of the cell cycle (20, 21). This is in favor of persistent R nucleic acid structures, comprising an DNA–RNA hybrid and a loops threatening genome integrity by impairing replication fork displaced single-stranded DNA (1). These structures also occur progression and/or causing DNA breaks, making the contribution naturally, mostly in a cotranscriptional manner and have been shown to play essential biological functions in different organ- Significance isms. Namely, R loops are essential for Escherichia coli plasmid replication, mitochondrial DNA replication, and human Ig class R loops form during transcription when the mRNA hybridizes switch recombination, and evidence accumulates supporting back to the template DNA forming a stable DNA–RNA hybrid. their involvement in DNA damage repair, transcription regula- Stable R loops can block replication and transcription machin- tion, and chromatin states in human cells and other organisms eries, leading to genome instability and human diseases. We – (2 5). Indeed, the presence of R loops in genomes has proven to report that the human mitochondrial degradosome (mtEXO), be more frequently detected than initially anticipated, as shown formed by SUV3 and PNPase, plays an important role in R-loop by the growing number of whole-genome sequencing studies metabolism in the mitochondria apart from preventing dsRNA mapping the genomic locations where these structures arise. accumulation, with implications in replication and preventing These studies dissecting the nature and distribution of R loops instability of the mitochondrial genome. Our findings help es- have helped establish common features aiding R-loop formation tablish a connection between the RNA processing activities of and stabilization, showing that negative supercoiling, GC con- the mtEXO and mitochondrial genome stability, affecting mito- tent, repetitive sequences, and the occurrence of nicks or breaks chondria homeostasis, cellular metabolism, and human diseases. on the DNA molecule are major contributing factors (6–10). Despite the specific cellular functions of certain R loops, these Author contributions: S.S. and A.A. designed research; S.S. and L.P.C. performed research; can alter the surrounding chromatin landscape and become an S.S. and A.A. analyzed data; and S.S. and A.A. wrote the paper. obstacle on the DNA molecule that can stall the replication The authors declare no conflict of interest. machinery and, subsequently, lead to DNA breaks, representing This article is a PNAS Direct Submission. U.B. is a guest editor invited by the Editorial an important source of DNA damage and a driver of genome Board. instability (11, 12). To regulate the accumulation of R loops, Published under the PNAS license. cells have developed different mechanisms that avoid or remove 1To whom correspondence should be addressed. Email: [email protected]. them. As an initial measure to avoid R-loop formation, RNA- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. binding proteins (RBPs) that associate with the transcription 1073/pnas.1807258115/-/DCSupplemental. machinery, such as the THO complex, bind the nascent RNA Published online October 9, 2018. 11024–11029 | PNAS | October 23, 2018 | vol. 115 | no. 43 www.pnas.org/cgi/doi/10.1073/pnas.1807258115 Downloaded by guest on October 2, 2021 of replication-associated DNA repair pathways in eliminating genomic instability by counteracting pathological R-loop accumu- them of paramount importance. lation and preventing replication–transcription conflicts, supporting The human mitochondrial DNA (mtDNA) is a small 16.6-kb the notion that this is a general mechanism for genome integrity. circular double-stranded DNA (dsDNA) molecule, containing 37 genes encoding essential components of the mitochondrial re- Results spiratory chain and specific tRNAs and rRNAs. Within each Loss of mtEXO Leads to Accumulation of DNA–RNA Hybrids and mitochondrion, several hundreds of mtDNA copies are present dsRNA in Mitochondria. A number of nuclear-encoded proteins and organized in mtDNA–protein complexes termed nucleoids involved in various aspects of DNA and RNA metabolism in- (22). mtDNA has a C-rich light (L) strand and a complementary cluding nuclear RNA surveillance, have been implicated in nu- G-rich heavy (H) strand, with one large noncoding region (NCR) clear R-loop prevention and R-loop–associated genome instability where most of the regulatory elements locate, including the H (11). To address whether these processes are conserved in non- strand origin of replication (OH), and the L and H strand pro- nuclear genomes, we analyzed whether mitochondrial RNA sur- moters (LSPs and HSPs, respectively). The NCR, or control region, veillance, in particular the mitochondrial degradosome (mtEXO), of many mtDNA molecules presents a three-stranded complex has any role in mitochondrial R-loop homeostasis and genome structure that contains a D loop formed by the 7S DNA, where integrity. To visualize and establish R-loop detection we relied on replication is initiated from the OH. Also at the control region, a immunofluorescence (IF) with the S9.6 anti-DNA–RNA hybrid stable R loop is found at high frequency, formed by a small RNA antibody and RNase H1 overexpression and/or in vitro treatment, transcribed from the LSP by the mitochondrial RNA poly- previously demonstrated to be bona fide readouts of R-loop merase (POLRMT) (23). This plays a critical role in mtDNA presence (6, 14, 15, 41). We found that silencing the mtEXO replication serving as a primer for leading-strand synthesis by exoribonuclease-encoding gene PNPT1 using siRNA (siPNPT1) γ the mitochondrial DNA polymerase Pol from OH, after pro- produced an unexpected punctate S9.6 staining pattern in the cessing by RNase H1 (19, 24). Both the D loop and R loop have cytoplasm. Importantly, depletion of the other mtEXO subunit, been implicated not only in replication of the mtDNA, but also the SUV3 helicase by siRNA (siSUV3), conferred the same in the organization and distribution of the mitochondrial ge- punctate
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