Dinucleotide Degradation by REXO2 Maintains Promoter Specificity in Mammalian Mitochondria.
ABSTRACT: Oligoribonucleases are conserved enzymes that degrade short RNA molecules of up to 5 nt in length and are assumed to constitute the final stage of RNA turnover. Here we demonstrate that REXO2 is a specialized dinucleotide-degrading enzyme that shows no preference between RNA and DNA dinucleotide substrates. A heart- and skeletal-muscle-specific knockout mouse displays elevated dinucleotide levels and alterations in gene expression patterns indicative of aberrant dinucleotide-primed transcription initiation. We find that dinucleotides act as potent stimulators of mitochondrial transcription initiation in vitro. Our data demonstrate that increased levels of dinucleotides can be used to initiate transcription, leading to an increase in transcription levels from both mitochondrial promoters and other, nonspecific sequence elements in mitochondrial DNA. Efficient RNA turnover by REXO2 is thus required to maintain promoter specificity and proper regulation of transcription in mammalian mitochondria.
Project description:Oligoribonucleases are conserved enzymes that degrade short RNA molecules of up to 5 nt in length, and are assumed to constitute the final stage of RNA turnover. We have investigated the function of the mammalian oligoribonuclease REXO2 through a combination of biochemical, structural and animal model studies. We find that REXO2 is a specialised dinucleotide degrading enzyme that shows no preference between RNA and DNA dinucleotide substrates. This specialisation is explained by crystal structures of REXO2 bound to RNA or DNA substrates. REXO2 is an essential gene, while a heart and skeletal muscle-specific knockout mouse displays elevated dinucleotide levels and alterations in gene expression patterns indicative of aberrant dinucleotide-primed transcription initiation. We find that dinucleotides act as potent stimulators of mitochondrial transcription initiation in vitro, and conclude that efficient RNA turnover is required to maintain the promoter specificity of transcription in mammalian mitochondria. Overall design: Total RNA was isolated from heart tissue from 52 week old control (Rexo2loxP/loxP) and Rexo2 knockout mice (Rexo2loxP/loxP, +/Ckmm), TruSeq libraries produced in triplicate, sequenced and analysed for differential expression. Mitochondrial RNA was isolated from heart tissue from 52 week old control (Rexo2loxP/loxP) and Rexo2 knockout mice (Rexo2loxP/loxP, +/Ckmm), PARE libraries produced in triplicate and sequenced for analysis of mitochondrial RNA processing.
Project description:RNA decay is a key element of mitochondrial RNA metabolism. To date, the only well-documented machinery that plays a role in mtRNA decay in humans is the complex of polynucleotide phosphorylase (PNPase) and SUV3 helicase, forming the degradosome. REXO2, a homolog of prokaryotic oligoribonucleases present in humans both in mitochondria and the cytoplasm, was earlier shown to be crucial for maintaining mitochondrial homeostasis, but its function in mitochondria has not been fully elucidated. In the present study, we created a cellular model that enables the clear dissection of mitochondrial and non-mitochondrial functions of human REXO2. We identified a novel mitochondrial short RNA, referred to as ncH2, that massively accumulated upon REXO2 silencing. ncH2 degradation occurred independently of the mitochondrial degradosome, strongly supporting the hypothesis that ncH2 is a primary substrate of REXO2. We also investigated the global impact of REXO2 depletion on mtRNA, revealing the importance of the protein for maintaining low steady-state levels of mitochondrial antisense transcripts and double-stranded RNA. Our detailed biochemical and structural studies provide evidence of sequence specificity of the REXO2 oligoribonuclease. We postulate that REXO2 plays dual roles in human mitochondria, 'scavenging' nanoRNAs that are produced by the degradosome and clearing short RNAs that are generated by RNA processing.
Project description:Human RNA exoribonuclease 2 (Rexo2) is an evolutionarily conserved 3'-to-5' DEDDh-family exonuclease located primarily in mitochondria. Rexo2 degrades small RNA oligonucleotides of <5 nucleotides (nanoRNA) in a way similar to Escherichia coli Oligoribonuclease (ORN), suggesting that it plays a role in RNA turnover in mitochondria. However, how Rexo2 preferentially binds and degrades nanoRNA remains elusive. Here, we show that Rexo2 binds small RNA and DNA oligonucleotides with the highest affinity, and it is most robust in degrading small nanoRNA into mononucleotides in the presence of magnesium ions. We further determined three crystal structures of Rexo2 in complex with single-stranded RNA or DNA at resolutions of 1.8-2.2 Å. Rexo2 forms a homodimer and interacts mainly with the last two 3'-end nucleobases of substrates by hydrophobic and ?-? stacking interactions via Leu53, Trp96, and Tyr164, signifying its preference in binding and degrading short oligonucleotides without sequence specificity. Crystal structure of Rexo2 is highly similar to that of the RNA-degrading enzyme ORN, revealing a two-magnesium-ion-dependent hydrolysis mechanism. This study thus provides the molecular basis for human Rexo2, showing how it binds and degrades nanoRNA into nucleoside monophosphates and plays a crucial role in RNA salvage pathways in mammalian mitochondria.
Project description:The Escherichia coli oligoribonuclease, ORN, has a 3' to 5' exonuclease activity specific for small oligomers that is essential for cell viability. The human homologue, REXO2, has hitherto been incompletely characterized, with only its in vitro ability to degrade small single-stranded RNA and DNA fragments documented. Here we show that the human enzyme has clear dual cellular localization being present both in cytosolic and mitochondrial fractions. Interestingly, the mitochondrial form localizes to both the intermembrane space and the matrix. Depletion of REXO2 by RNA interference causes a strong morphological phenotype in human cells, which show a disorganized network of punctate and granular mitochondria. Lack of REXO2 protein also causes a substantial decrease of mitochondrial nucleic acid content and impaired de novo mitochondrial protein synthesis. Our data constitute the first in vivo evidence for an oligoribonuclease activity in human mitochondria.
Project description:Pheochromocytoma (PCC) is a rare, mostly benign tumour of the adrenal medulla. Hereditary PCC accounts for ~35% of cases and has been associated with germline mutations in several cancer susceptibility genes (e.g., KIF1B, SDHB, VHL, SDHD, RET). We performed whole-exome sequencing in a family with four PCC-affected patients in two consecutive generations and identified a potential novel candidate pathogenic variant in the REXO2 gene that affects splicing (c.531-1G>T (NM 015523.3)), which co-segregated with the phenotype in the family. REXO2 encodes for RNA exonuclease 2 protein and localizes to 11q23, a chromosomal region displaying allelic imbalance in PCC. REXO2 protein has been associated with DNA repair, replication and recombination processes and thus its inactivation may contribute to tumorigenesis. While the study suggests that this novel REXO2 gene variant underlies PCC in this family, additional functional studies are required in order to establish the putative role of the REXO2 gene in PCC predisposition.
Project description:Investigation of the role of human REXO2 in mitochondrial RNA metabolism. Overall design: The dataset corresponds to RNAseq studies and comprises experiment performed in triplicate, with the exception of REXO2 silencing sample for which duplicate was analyzed. The aim of this study was to examine the influence of REXO2 silencing on mitochondrial transcriptome. To this end we engineered two stable cell lines with the use of human HeLa Flp-In T-Rex cells as parental. First cell line inducible expressed miRNAs silencing endogenous copy of REXO2 gene while second one expressed additionally transgenic version of REXO2 which contained silent mutations causing insensitivity to miRNA. We also analyzed RNA isolated from parental HeLa Flp-In T-Rex cells. RNAseq libraries were prepared with the use of strand-specific library preparation procedures. RNAs were random fragmented and reverse transcribed using random oligomers as primers (dUTP-based protocol, see PMID: 29590189, PMID: 22609201; this pipeline enables analysis of RNAs (> ~100 nucleotides)). RNA was isolated from unfractionated cells using TRI-Reagent. Before preparation of the libraries total RNA was subjected to depletion of nuclear-encoded rRNAs (Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat), Epicenter). Libraries were sequenced with the help of Illumina sequencing platform.
Project description:Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non-canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts, and up to ~15% NAD+ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD+ and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial transcriptional outputs, sensing NAD+ and NADH levels and adjusting transcriptional outputs accordingly.
Project description:Human Nudt16 (hNudt16) is a member of the Nudix family of hydrolases, comprising enzymes catabolizing various substrates including canonical (d)NTPs, oxidized (d)NTPs, nonnucleoside polyphosphates, and capped mRNAs. Decapping activity of the Xenopus laevis (X29) Nudt16 homolog was observed in the nucleolus, with a high specificity toward U8 snoRNA. Subsequent studies have reported cytoplasmic localization of mammalian Nudt16 with cap hydrolysis activity initiating RNA turnover, similar to Dcp2. The present study focuses on hNudt16 and its hydrolytic activity toward dinucleotide cap analogs and short capped oligonucleotides. We performed a screening assay for potential dinucleotide and oligonucleotide substrates for hNudt16. Our data indicate that dinucleotide cap analogs and capped oligonucleotides containing guanine base in the first transcribed nucleotide are more susceptible to enzymatic digestion by hNudt16 than their counterparts containing adenine. Furthermore, unmethylated dinucleotides (GpppG and ApppG) and respective oligonucleotides (GpppG-16nt and GpppA-16nt) were hydrolyzed by hNudt16 with greater efficiency than were m7GpppG and m7GpppG-16nt. In conclusion, we found that hNudt16 hydrolysis of dinucleotide cap analogs and short capped oligonucleotides displayed a broader spectrum specificity than is currently known.
Project description:The light strand promoter of mammalian mitochondrial DNA gives rise to a primary transcript, but also to the RNA primer necessary for initiation of replication and 7S DNA synthesis as well as 7S RNA. Here we have studied the turnover of 7S DNA in isolated rat liver mitochondria and whether import of mitochondrial transcription factor A (mtTFA), which is necessary for transcription initiation, increases its rate of synthesis. 7S DNA was present as two species, probably due to two different sites of RNA-DNA transition. Time course and pulse-chase experiments showed that the half-life of this DNA is approximately 45 min. Import of mtTFA, produced in vitro, into the mitochondrial matrix in stoichiometric amounts significantly increased the rate of 7S DNA formation. We conclude that isolated rat liver mitochondria faithfully synthesize and degrade 7S DNA and that increased matrix levels of mtTFA are sufficient to increase its rate of synthesis, strongly supporting the hypothesis that this process is transcription primed.