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Antisense oligonucleotide (ASO) drugs that trigger RNase H1 cleavage of target RNAs have been developed to treat various diseases. Basic pharmacological principles suggest that the development of tolerance is a common response to pharmacological interventions. In this manuscript, for the first time we report a molecular mechanism of tolerance that occurs with some ASOs. Two observations stimulated our interest: some RNA targets are difficult to reduce with RNase H1 activating ASOs and some ASOs display a shorter duration of activity than the prolonged target reduction typically observed. We found that certain ASOs targeting the coding region of some mRNAs that initially reduce target mRNAs can surprisingly increase the levels of the corresponding pre-mRNAs. The increase in pre-mRNA is delayed and due to enhanced transcription and likely also slower processing. This process requires that the ASOs bind in the coding region and reduce the target mRNA by RNase H1 while the mRNA resides in the ribosomes. The pre-mRNA increase is dependent on UPF3A and independent of the NMD pathway or the XRN1-CNOT pathway. The response is consistent in multiple cell lines and independent of the methods used to introduce ASOs into cells.

The rapid RNase H1-dependent mislocalization of heterodimer proteins P54nrb and PSF to nucleoli is an early event in the pathway that explains the effects of most toxic phosphorothioate ASOs (PS-ASOs). Using a recently developed NanoLuciferace (NLuc)-based structural complementation reporter system which allows us to observe ASO/protein interactions in real time in live cells, we have determined that safe and toxic PS-ASOs associate with these proteins with kinetics and impact on subcellular localization that differ. Toxic PS-ASOs interact in a complex that includes RNase H1, P54nrb and PSF; but RNase H1/P54nrb complexes were observed in only the cells treated with toxic, but not safe PS-ASOs. In addition, experiments performed in vitro suggest that RNA is also a required component of the complex. The protein–protein interaction between P54nrb and RNase H1 requires the spacer region of RNAse H1, while the P54nrb core domains are required for association with RNase H1. In addition, we have determined that PS-ASOs bind P54nrb via RRM1 and RRM2, while they bind RNase H1 primarily via the hybrid binding domain, however catalytic domain interactions also contribute to overall affinity. These ASO–protein interactions are highly influenced by the chemistry of the PS-ASO binding environment, however little correlation between affinity for specific proteins and PS-ASO toxicity was observed.

Replication of sufficient mitochondrial DNA (mtDNA) is essential for maintaining mitochondrial functions in mammalian cells. During mtDNA replication, RNA primers must be removed before the nascent circular DNA strands rejoin. This process involves mitochondrial RNase H1, which removes most of the RNA primers but leaves two ribonucleotides attached to the 5′ end of nascent DNA. A subsequent 5′-exonuclease is required to remove the residual ribonucleotides, however, it remains unknown if any mitochondrial 5′-exonuclease could remove two RNA nucleotides from a hybrid duplex DNA. Here, we report that human mitochondrial Exonuclease G (ExoG) may participate in this particular process by efficiently cleaving at RNA–DNA junctions to remove the 5′-end RNA dinucleotide in an RNA/DNA hybrid duplex. Crystal structures of human ExoG bound respectively with DNA, RNA/DNA hybrid and RNA–DNA chimeric duplexes uncover the underlying structural mechanism of how ExoG specifically recognizes and cleaves at RNA–DNA junctions of a hybrid duplex with an A-form conformation. This study hence establishes the molecular basis of ExoG functioning as a unique 5′-exonuclease to mediate the flap-independent RNA primer removal process during mtDNA replication to maintain mitochondrial genome integrity.

DEAR EDITOR, The feasibility of testing multiple genes at once using Next Generation Sequencing, particularly Whole Exome Sequencing (WES), has expanded the phenotypic spectrum associated with many disease genes. Distinguishing atypical presentation from combined gene effects and incidental from causal findings can however be challenging, particularly when composite phenotypes are themselves extremely rare. [...]

Removal of ribonucleotides (rNMPs) incorporated into the genome by the ribonucleotide excision repair (RER) is essential to avoid genetic instability. In eukaryotes, the RNaseH2 is the only known enzyme able to incise 5′ of the rNMP, starting the RER process, which is subsequently carried out by replicative DNA polymerases (Pols) δ or ϵ, together with Flap endonuclease 1 (Fen-1) and DNA ligase 1. Here, we show that the DEAD-box RNA helicase DDX3X has RNaseH2-like activity and can support fully reconstituted in vitro RER reactions, not only with Pol δ but also with the repair Pols β and λ. Silencing of DDX3X causes accumulation of rNMPs in the cellular genome. These results support the existence of alternative RER pathways conferring high flexibility to human cells in responding to the threat posed by rNMPs incorporation.

Long interspersed nuclear element 1 is an autonomous non-long terminal repeat retrotransposon that comprises ∼17% of the human genome. Its spontaneous retrotransposition and the accumulation of heritable L1 insertions can potentially result in genome instability and sporadic disorders. Moloney leukemia virus 10 homolog (MOV10), a putative RNA helicase, has been implicated in inhibiting L1 replication, although its underlying mechanism of action remains obscure. Moreover, the physiological relevance of MOV10-mediated L1 regulation in human disease has not yet been examined. Using a proteomic approach, we identified RNASEH2 as a binding partner of MOV10. We show that MOV10 interacts with RNASEH2, and their interplay is crucial for restricting L1 retrotransposition. RNASEH2 and MOV10 co-localize in the nucleus, and RNASEH2 binds to L1 RNAs in a MOV10-dependent manner. Small hairpin RNA-mediated depletion of either RNASEH2A or MOV10 results in an accumulation of L1-specific RNA-DNA hybrids, suggesting they contribute to prevent formation of vital L1 heteroduplexes during retrotransposition. Furthermore, we show that RNASEH2-MOV10-mediated L1 restriction downregulates expression of the rheumatoid arthritis-associated inflammatory cytokines and matrix-degrading proteinases in synovial cells, implicating a potential causal relationship between them and disease development in terms of disease predisposition.

RNA sequencing (RNA-seq) is extensively used to quantify gene expression transcriptome-wide. Although often paired with polyadenylate (poly(A)) selection to enrich for messenger RNA (mRNA), many applications require alternate approaches to counteract the high proportion of ribosomal RNA (rRNA) in total RNA. Recently, digestion using RNaseH and antisense DNA oligomers tiling target rRNAs has emerged as an alternative to commercial rRNA depletion kits. Here, we present a streamlined, more economical RNaseH-mediated rRNA depletion with substantially lower up-front costs, using shorter antisense oligos only sparsely tiled along the target RNA in a 5-min digestion reaction. We introduce a novel Web tool, Oligo-ASST, that simplifies oligo design to target regions with optimal thermodynamic properties, and additionally can generate compact, common oligo pools that simultaneously target divergent RNAs, e.g. across different species. We demonstrate the efficacy of these strategies by generating rRNA-depletion oligos for Xenopus laevis and for zebrafish, which expresses two distinct versions of rRNAs during embryogenesis. The resulting RNA-seq libraries reduce rRNA to5% of aligned reads, on par with poly(A) selection, and also reveal expression of many non-adenylated RNA species. Oligo-ASST is freely available at https://mtleelab.pitt.edu/oligo to design antisense oligos for any taxon or to target any abundant RNA for depletion.

Influenza A virus (IAV) genomes are composed of eight single-stranded RNA segments that are coated by viral nucleoprotein (NP) molecules. Classically, the interaction between NP and viral RNA (vRNA) is depicted as a uniform pattern of ‘beads on a string’. Using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP), we identified the vRNA binding profiles of NP for two H1N1 IAV strains in virions. Contrary to the prevailing model for vRNA packaging, NP does not bind vRNA uniformly in the A/WSN/1933 and A/California/07/2009 strains, but instead each vRNA segment exhibits a unique binding profile, containing sites that are enriched or poor in NP association. Intriguingly, both H1N1 strains have similar yet distinct NP binding profiles despite extensive sequence conservation. Peaks identified by HITS-CLIP were verified as true NP binding sites based on insensitivity to DNA antisense oligonucleotide-mediated RNase H digestion. Moreover, nucleotide content analysis of NP peaks revealed that these sites are relatively G-rich and U-poor compared to the genome-wide nucleotide content, indicating an as-yet unidentified sequence bias for NP association in vivo. Taken together, our genome-wide study of NP–vRNA interaction has implications for the understanding of influenza vRNA architecture and genome packaging.

Branchpoint nucleotides of intron lariats induce pausing of DNA synthesis by reverse transcriptases (RTs), but it is not known yet how they direct RT RNase H activity on branched RNA (bRNA). Here, we report the effects of the two arms of bRNA on branchpoint-directed RNA cleavage and mutation produced by Moloney murine leukemia virus (M-MLV) RT during DNA polymerization. We constructed a long-chained bRNA template by splinted-ligation. The bRNA oligonucleotide is chimeric and contains DNA to identify RNA cleavage products by probe hybridization. Unique sequences surrounding the branchpoint facilitate monitoring of bRNA purification by terminal-restriction fragment length polymorphism analysis. We evaluate the M-MLV RT-generated cleavage and mutational patterns. We find that cleavage of bRNA and misprocessing of the branched nucleotide proceed arm-specifically. Bypass of the branchpoint from the 2΄-arm causes single-mismatch errors, whereas bypass from the 3΄-arm leads to deletion mutations. The non-template arm is cleaved when reverse transcription is primed from the 3΄-arm but not from the 2΄-arm. This suggests that RTs flip ∼180° at branchpoints and RNases H cleave the non-template arm depending on its accessibility. Our observed interplay between M-MLV RT and bRNA would be compatible with a bRNA-mediated control of retroviral and related retrotransposon replication.

Antisense oligonucleotides (ASOs) are most commonly designed to reduce targeted RNA via RNase H1-dependent degradation, however kinetic parameters for ASO-mediated targeting and subsequent cleavage and degradation of RNA in living cells are poorly understood. In this manuscript we use an inducible minigene system to determine the time course of ASO activity in the cell. Estimates of the time required for the ASO to enter and traverse the cell, scan the target mRNA, bind the cognate site, recruit RNase H1 and initiate cleavage, are presented in the context of transcription and mRNA processing rates. Data are also presented which indicate that rates for RNase H1-dependent ASO-mediated degradation of the targeted RNAs are different for nuclear-retained versus RNAs exported to the cytoplasm and that the level of RNase H1 in the cell and cellular compartments is limiting to the rate of ASO activity. In both cellular compartments RNase H1 ASOs essentially double the endogenous rates of clearance of the target RNA. Overexpression of Escherichia coli RNase H1 or the presence of multiple cognate sites each further increase the rate of target RNA degradation.

Nucleases play important roles in nucleic acid processes, such as replication, repair and recombination. Recently, we identified a novel single-strand specific 3′-5′ exonuclease, PfuExo I, from the hyperthermophilic archaeon Pyrococcus furiosus, which may be involved in the Thermococcales-specific DNA repair system. PfuExo I forms a trimer and cleaves single-stranded DNA at every two nucleotides. Here, we report the structural basis for the cleavage mechanism of this novel exonuclease family. A structural analysis of PhoExo I, the homologous enzyme from P. horikoshii OT3, showed that PhoExo I utilizes an RNase H-like active site and possesses a 3′-OH recognition site ∼9 A away from the active site, which enables cleavage at every two nucleotides. Analyses of the heterotrimeric and monomeric PhoExo I activities showed that trimerization is indispensable for its processive cleavage mechanism, but only one active site of the trimer is required.

Ribonuclease H (RNase H) specifically degrades the RNA of RNA/DNA hybrid. Recent study has shown that a single ribonucleotide is embedded in DNA double strand at every few thousand base pairs in human genome, and human RNase H2 is involved in its removal. Here, we examined the effects of neutral salts and pH on the activity and stability of human RNase H2. NaCl, KCl, RbCl and NaBr increased the activity to 170-390% at 10-60 mM, while LiCl, LiBr and CsCl inhibited it, suggesting that species of cation, but not anion, is responsible for the effect on activity. NaCl and KCl increased the stability by decreasing the first-order rate constant of the inactivation to 50-60% at 60-80 mM. The activity at 25-35 °C exhibited a narrow bell-shaped pH-dependence with the acidic and alkaline pKe (pKe1 and pKe2) values of 7.3 - 7.6 and 8.1 - 8.8, respectively. Enthalpy changes (ΔH°) of deprotonation were 5 ± 21 kJ mol-1 for pKe1 and 68 ± 25 kJ mol-1 for pKe2. These results suggest that the ionizable groups responsible for pKe1 may be two out of Asp34, Glu35 and Asp141 of DEDD motif, and that for pKe2 may be Lys69 of DSK motif.

Ribonucleotides (rNs) incorporated in the genome by DNA polymerases (Pols) are removed by RNase H2. Cytidine and guanosine preferentially accumulate over the other rNs. Here we show that human Pol η can incorporate cytidine monophosphate (rCMP) opposite guanine, 8-oxo-7,8-dihydroguanine, 8-methyl-2΄-deoxyguanosine and a cisplatin intrastrand guanine crosslink (cis-PtGG), while it cannot bypass a 3-methylcytidine or an abasic site with rNs as substrates. Pol η is also capable of synthesizing polyribonucleotide chains, and its activity is enhanced by its auxiliary factor DNA Pol δ interacting protein 2 (PolDIP2). Human RNase H2 removes cytidine and guanosine less efficiently than the other rNs and incorporation of rCMP opposite DNA lesions further reduces the efficiency of RNase H2. Experiments with XP-V cell extracts indicate Pol η as the major basis of rCMP incorporation opposite cis-PtGG. These results suggest that translesion synthesis by Pol η can contribute to the accumulation of rCMP in the genome, particularly opposite modified guanines., Nucleic Acids Research, 45 (5), ISSN:1362-4962, ISSN:0301-5610

Aicardi-Goutières syndrome (AGS) is an inflammatory encephalopathy caused by defective nucleic acids metabolism. Over 50% of AGS mutations affect RNase H2 the only enzyme able to remove single ribonucleotidemonophosphates (rNMPs) embedded in DNA. Ribonucleotide triphosphates (rNTPs) are incorporated into genomic DNA with relatively high frequency during normal replication making DNA more susceptible to strand breakage and mutations. Here we demonstrate that human cells depleted of RNase H2 show impaired cell cycle progression associated with chronic activation of post-replication repair (PRR) and genome instability. We identify a similar phenotype in cells derived from AGS patients, which indeed accumulate rNMPs in genomic DNA and exhibit markers of constitutive PRR and checkpoint activation. Our data indicate that in human cells RNase H2 plays a crucial role in correcting rNMPs misincorporation, preventing DNA damage. Such protective function is compromised in AGS patients and may be linked to unscheduled immune responses. These findings may be relevant to shed further light on the mechanisms involved in AGS pathogenesis.

7,8-Dihydro-8-oxo-deoxyguanosine (8oxodG) is a highly premutagenic DNA lesion due to its ability to mispair with adenine. Schizosaccharomyces pombe lacks homologs for relevant enzymes that repair 8oxodG, which suggests that this lesion could be persistent and must be tolerated. Here we show that SpPol4, the unique PolX in fission yeast, incorporates ATP opposite 8oxodG almost exclusively when all nucleotides (ribos and deoxys) are provided at physiological concentrations. Remarkably, this SpPol4-specific reaction could also occur during the NHEJ of DSBs. In cell extracts, misincorporation of ATP opposite 8oxodG was shown to be SpPol4-specific, although RNase H2 efficiently recognized the 8oxodG:AMP mispair to remove AMP and trigger error-free incorporation of dCTP. These data are the first evidence that ribonucleotides can be used safely for 8oxodG tolerance, suggesting that insertion of the highly abundant ATP substrate could be beneficial to promote efficient and error-free repair of 8oxodG-associated DSBs. Moreover, we demonstrate that purified SpPol4 uses 8oxo-dGTP and 8oxo-GTP as substrates for DNA polymerization, although with poor efficiency compared to the incorporation of undamaged nucleotides opposite either 8oxodG or undamaged templates. This suggests that SpPol4 is specialized in tolerating 8oxodG as a DNA template, without contributing significantly to the accumulation of this lesion in the DNA.

HIV-1 reverse transcriptase (RT), a critical enzyme 10 of the HIV life cycle and an important drug target, undergoes complex and largely uncharacterized conformational rearrangements that underlie its asymmetric folding, dimerization and subunit-selective ribonuclease H domain (RH) proteolysis. In the 15 present article we have used a combination of NMR spectroscopy, small angle X-ray scattering and X-ray crystallography to characterize the p51 and p66 monomers and the conformational maturation of the p66/p66 0 homodimer. The p66 monomer exists as a 20 loosely structured molecule in which the fingers/ palm/connection, thumb and RH substructures are connected by flexible (disordered) linking segments. The initially observed homodimer is asymmetric and includes two fully folded RH domains, while exhibiting 25 other conformational features similar to that of the RT heterodimer. The RH 0 domain of the p66 0 subunit undergoes selective unfolding with time constant ! 6.5 h, consistent with destabilization due to residue transfer to the polymerase 0 domain on the 30 p66 0 subunit. A simultaneous increase in the intensity of resonances near the random coil positions is characterized by a similar time constant. Consistent with the residue transfer hypothesis, a construct of the isolated RH domain lacking the two N-terminal 35 residues is shown to exhibit reduced stability. These results demonstrate that RH 0 unfolding is coupled to homodimer formation.

We analyzed a multi-drug resistant (MR) HIV-1 reverse transcriptase (RT), subcloned from a patient-derived subtype CRF02_AG, harboring 45 amino acid exchanges, amongst them four thymidine analog mutations (TAMs) relevant for high-level AZT (azidothymidine) resistance by AZTMP excision (M41L, D67N, T215Y, K219E) as well as four substitutions of the AZTTP discrimination pathway (A62V, V75I, F116Y and Q151M). In addition, K65R, known to antagonize AZTMP excision in HIV-1 subtype B was present. Although MR-RT harbored the most significant amino acid exchanges T215Y and Q151M of each pathway, it exclusively used AZTTP discrimination, indicating that the two mechanisms are mutually exclusive and that the Q151M pathway is obviously preferred since it confers resistance to most nucleoside inhibitors. A derivative was created, additionally harboring the TAM K70R and the reversions M151Q as well as R65K since K65R antagonizes excision. MR-R65K-K70R-M151Q was competent of AZTMP excision, whereas other combinations thereof with only one or two exchanges still promoted discrimination. To tackle the multi-drug resistance problem, we tested if the MR-RTs could still be inhibited by RNase H inhibitors. All MR-RTs exhibited similar sensitivity toward RNase H inhibitors belonging to different inhibitor classes, indicating the importance of developing RNase H inhibitors further as anti-HIV drugs.

7 páginas, 6 figuras, 1 tabla., To get further insight into the effect that THO/TREX and R-loops have in transcription-associated recombination and transcription, we analyzed the ability to form R-loops of hpr1-101, a THO mutation that impairs transcription and mRNP biogenesis without triggering hyper-recombination. Human AID, a cytidine deaminase that acts on ssDNA displaced by RNA-DNA hybrids, strongly induced both hyper-recombination and hyper-mutation in hpr1-101, similar to hpr1Δ mutants. However, in contrast to hpr1Δ, AID-induced mutations in hpr1-101 occur at similar frequencies in both the transcribed and non-transcribed strands, implying that the enhanced AID action in these mutants is not caused by co-transcriptional R-loops. These results indicate for the first time that THO has a transcriptional function that is not mediated by R-loops, providing a new perspective for the understanding of the coupling of transcription with mRNP biogenesis and export., Spanish Ministry of Science and Innovation (BFU2006-05260, CSD2007-015); Junta de Andalucía (CVI-2549, BIO-102). B.G.-G. was a recipient of a predoctoral training grant from the Spanish Ministry of Science and Innovation. Funding for open access charge: grant BIO-102 from Junta de Andalucía.

While much is known about abasic DNA, the biological impact of abasic RNA is largely unexplored. To test the mutagenic potential of this RNA lesion in the context of retroviruses, we synthesized a 31-mer oligoribonucleotide containing an abasic (rAS) site and used it as a template for studying DNA primer extension by HIV-1, avian myeloblastosis virus (AMV) and moloney murine leukemia virus (MMLV) reversed transcriptases (RT). We found that trans-lesion synthesis readily takes place with HIV-1 RT and to a lesser extent with AMV RT while MMLV RT aborts DNA synthesis. The preference of dNTP incorporation follows the order A approximately GC approximately T and thus obeys to the 'A-rule'. In the case of HIV-1 RT, we measured the kinetic data of dNTP incorporation and compared it to abasic DNA. We found that A-incorporation is only 2-fold slower relative to a matched (undamaged) RNA template while it is 7-fold slower in the case of DNA. Furthermore, there is less discrimination in incorporation between the four dNTPs in the case of abasic RNA compared to abasic DNA. These experiments clearly point to a higher promiscuity of lesion bypass on abasic RNA. Given their known higher chemical stability, such rAS sites can clearly contribute to (retro)viral evolution.

HIV-1 reverse transcriptase (RT) connection subdomain mutations at positions 348, 369 and 376 have been associated with resistance to non-nucleoside RT inhibitors (NNRTIs). N348I may interfere with the initiation of (+)-strand DNA synthesis by reducing polypurine tract (PPT) removal in the presence of nevirapine. The effect of NNRTIs on the RNase H-mediated cleavage of PPT-containing template-primers has been studied with wild-type HIV-1 RT and mutants N348I, T369I, T369V, T376S and N348I/T369I. In the presence of NNRTIs, all RTs were able to stimulate PPT cleavage after primer elongation. The enhancing effects of nevirapine and efavirenz were reduced in RTs carrying mutation N348I, and specially N348I/T369I. However, those mutations had no effect on rilpivirine-mediated cleavage. Prior to elongation, the PPT remains resilient to cleavage, although efavirenz and rilpivirine facilitate RNase H-mediated trimming of its 3′-end. The integrity of the 3′-end is essential for the initiation of (+)-strand DNA synthesis. In the presence of dNTPs, rilpivirine was the most effective inhibitor of (+)-strand DNA synthesis blocking nucleotide incorporation and preventing usage of available PPT primers. The N348I/T369I RT showed reduced ability to generate short RNA products revealing a cleavage window defect. Its lower RNase H activity could be attributed to enhanced rigidity compared to the wild-type enzyme., The Spanish Ministry of Economy and Competitiveness [BIO2010/15542 and BIO2013-48788-C2-1-R (to L.M.-A.); SAF2013-41421-R (to M.A.M.)]; the Spanish Ministry of Health, Social Services and Equality [EC11-025]; the Fundación Ramón Areces, Madrid, Spain (institutional grant to CBMSO). Funding for open access charge:The Spanish Ministry of Economy and Competitiveness[BIO2013-48788-C2-1-R].

Plant mitochondrial group II introns do not all possess hallmark ribozymic features such as the bulged adenosine involved in lariat formation. To gain insight into their splicing pathways, we have examined the physical form of excised introns in germinating wheat embryos. Using RT-PCR and cRT-PCR, we observed conventional lariats consistent with a two-step transesterification pathway for introns such as nad2 intron 4, but this was not the case for the cox2 intron or nad1 intron 2. For cox2, we detected full-length linear introns, which possess non-encoded 3'terminal adenosines, as well as heterogeneous circular introns, which lack 3' nucleotide stretches. These observations are consistent with hydrolytic splicing followed by polyadenylation as well as an in vivo circularization pathway, respectively. The presence of both linear and circular species in vivo is supported by RNase H analysis. Furthermore, the nad1 intron 2, which lacks a bulged nucleotide at the branchpoint position, comprised a mixed population of precisely full-length molecules and circular ones which also include a short, discrete block of non-encoded nucleotides. The presence of these various linear and circular forms of excised intron molecules in plant mitochondria points to multiple novel group II splicing mechanisms in vivo.

Ribonucleases H have mostly been implicated in eliminating short RNA primers used for initiation of lagging strand DNA synthesis. Escherichia coli RNase HI cleaves these RNA–DNA hybrids in a distributive manner. We report here that eukaryotic RNases H1 have evolved to be processive enzymes by attaching a duplex RNA-binding domain to the RNase H region. Highly conserved amino acids of the duplex RNA-binding domain are required for processivity and nucleic acid binding, which leads to dimerization of the protein. The need for a processive enzyme underscores the importance in eukaryotic cells of processing long hybrids, most of which remain to be identified. However, long RNA–DNA hybrids formed during immunoglobulin class-switch recombination are potential targets for RNase H1 in the nucleus. In mitochondria, where RNase H1 is essential for DNA formation during embryogenesis, long hybrids may be involved in DNA replication.

Asp443 and Glu478 are essential active site residues in the RNase H domain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). We have investigated the effects of substituting Asn for Asp443 or Gln for Glu478 on the fidelity of DNA-dependent DNA synthesis of phylogenetically diverse HIV-1 RTs. In M13mp2 lacZα-based forward mutation assays, HIV-1 group M (BH10) and group O RTs bearing substitutions D443N, E478Q, V75I/D443N or V75I/E478Q showed 2.0-to 6.6-fold increased accuracy in comparison with the corresponding wild-type enzymes. This was a consequence of their lower base substitution error rates. One-nucleotide deletions and insertions represented between 30 and 68% of all errors identified in the mutational spectra of RNase H-deficient HIV-1 group O RTs. In comparison with the wild-type RT, these enzymes showed higher frameshift error rates and higher dissociation rate constants (koff) for DNA/DNA template-primers. The effects on frameshift fidelity were similar to those reported for mutation E89G and suggest that in HIV-1 group O RT, RNase H inactivation could affect template/primer slippage. Our results support a role for the RNase H domain during plus-strand DNA polymerization and suggest that mutations affecting RNase H function could also contribute to retrovirus variability during the later steps of reverse transcription. © 2013 The Author(s)., Ministry of Economy and Competitiveness (Spain) [BIO2010/15542]; Ministry of Health, Social Services and Equality (Spain) [EC11-025]; Fondo de Investigacion Sanitaria [RD06/0006]; Fundacion Ramon Areces

Helicase–nuclease systems dedicated to DNA end resection in preparation for homologous recombination (HR) are present in all kingdoms of life. In thermophilic archaea, the HerA helicase and NurA nuclease cooperate with the highly conserved Mre11 and Rad50 proteins during HR-dependent DNA repair. Here we show that HerA and NurA must interact in a complex with specific subunit stoichiometry to process DNA ends efficiently. We determine crystallographically that NurA folds in a toroidal dimer of intertwined RNaseH-like domains. The central channel of the NurA dimer is too narrow for double-stranded DNA but appears well suited to accommodate one or two strands of an unwound duplex. We map a critical interface of the complex to an exposed hydrophobic epitope of NurA abutting the active site. Based upon the presented evidence, we propose alternative mechanisms of DNA end processing by the HerA-NurA complex.

Background: Dnmt3L, a member of the DNA methyltransferase 3 family, lacks enzymatic activity but is required for de-novo methylation of imprinted genes in oocytes and for transposon repression in male germ cells. Methods: We used northern blots, RT-PCR, 5' rapid amplification of complementary DNA (cDNA) ends (RACE), RNase H mapping, real-time/quantitative RT-PCR and in situ hybridization to identify and characterize Dnmt3L transcripts produced during germ cell development. Results: Mouse Dnmt3L uses three sex-specific promoters, not the single promoter previously thought. A promoter active in prospermatogonia drives transcription of an mRNA encoding the full-length protein in perinatal testis, where de-novo methylation occurs. Late pachytene spermatocytes activate a second promoter in intron 9 of the Dnmt3L gene. After this stage, the predominant transcripts are three truncated mRNAs, which appear to be non-coding. We could also detect similar adult testis transcripts in humans. In the mouse ovary, an oocyte-specific promoter located in an intron of the neighbouring autoimmune regulator (Aire) gene produces a transcript with the full open reading frame (ORF). This is the only Dnmt3L transcript found in growing oocytes and is absent in the oocytes of Dnmt3L-/- females. Conclusions: Sex-specific promoters control Dnmt3L expression in the mouse germ line, mirroring the situation at the Dnmt1 and Dnmt3A loci. Canadian Institutes of Health Research Northern Ireland HPSS Cancer Royal Society NIH Vice-Chancellor’s Research studentship CIHR studentship William Dawson Scholar of McGill University Scholar of the Fonds de la recherche en santé du Quebec BBSRC

The composition of RNase H2 has been a long-standing problem. Whereas bacterial and archaeal RNases H2 are active as single polypeptides, the Saccharomyces cerevisiae homolog, Rnh2Ap, when expressed in Escherichia coli, fails to produce an active RNase H2. By affinity chromatography purification and identification of polypeptides associated with a tagged S.cerevisiae Rnh2Ap, we obtained a complex of three proteins [Rnh2Ap (Rnh201p), Ydr279p (Rnh202p) and Ylr154p (Rnh203p)] that together are necessary and sufficient for RNase H2 activity [correction]. Deletion of the gene encoding any one of the proteins or mutations in the catalytic site in Rnh2A led to loss of RNase H2 activity. Even when S.cerevisiae RNase H2 is catalytically compromised, it still exhibits a preference for cleavage of the phosphodiester bond on the 5' side of a ribonucleotide-deoxyribonucleotide sequence in substrates mimicking RNA-primed Okazaki fragments or a single ribonucleotide embedded in a duplex DNA. Interestingly, Ydr279p and Ylr154p have homologous proteins only in closely related species. The multisubunit nature of S.cerevisiae RNase H2 may be important both for structural purposes and to provide a means of interacting with other proteins involved in DNA replication/repair and transcription.

The key step in bacterial translation is formation of the pre-initiation complex. This requires initial contacts between mRNA, fMet-tRNA and the 30S subunit of the ribosome, steps that limit the initiation of translation. Here we report a method for improving translational initiation, which allows expression of several previously non-expressible genes. This method has potential applications in heterologous protein synthesis and high-throughput expression systems. We introduced a synthetic RNA stem-loop (stem length, 7 bp; DeltaG(0) = -9.9 kcal/mol) in front of various gene sequences. In each case, the stem-loop was inserted 15 nt downstream from the start codon. Insertion of the stem-loop allowed in vitro expression of five previously non-expressible genes and enhanced the expression of all other genes investigated. Analysis of the RNA structure proved that the stem-loop was formed in vitro, and demonstrated that stabilization of the ribosome binding site is due to stem-loop introduction. By theoretical RNA structure analysis we showed that the inserted RNA stem-loop suppresses long-range interactions between the translation initiation domain and gene-specific mRNA sequences. Thus the inserted RNA stem-loop supports the formation of a separate translational initiation domain, which is more accessible to ribosome binding.

The antibiotic chloramphenicol produces modifications in 23S rRNA when bound to ribosomes from the bacterium Escherichia coli and the archaeon Halobacterium halobium and irradiated with 365 nm light. The modifications map to nucleotides m(5)U747 and C2611/C2612, in domains II and V, respectively, of E.coli 23S rRNA and G2084 (2058 in E.coli numbering) in domain V of H.halobium 23S rRNA. The modification sites overlap with a portion of the macrolide binding site and cluster at the entrance to the peptide exit tunnel. The data correlate with the recently reported chloramphenicol binding site on an archaeal ribosome and suggest that a similar binding site is present on the E.coli ribosome.

Human RNase H1 binds double-stranded RNA via its N-terminal domain and RNA-DNA hybrid via its C-terminal RNase H domain, the latter being closely related to Escherichia coli RNase HI. Using SELEX, we have generated a set of DNA sequences that can bind efficiently (K(d) values ranging from 10 to 80 nM) to the human RNase H1. None of them could fold into a simple perfect double-stranded DNA hairpin confirming that double-stranded DNA does not constitute a trivial ligand for the enzyme. Only two of the 37 DNA aptamers selected were inhibitors of human RNase H1 activity. The two inhibitory oligomers, V-2 and VI-2, were quite different in structure with V-2 folding into a large, imperfect but stable hairpin loop. The VI-2 structure consists of a central region unimolecular quadruplex formed by stacking of two guanine quartets flanked by the 5' and 3' tails that form a stem of six base pairs. Base pairing between the 5' and 3' tails appears crucial for conferring the inhibitory properties to the aptamer. Finally, the inhibitory aptamers were capable of completely abolishing the action of an antisense oligonucleotide in a rabbit reticulocyte lysate supplemented with human RNase H1, with IC50 ranging from 50 to 100 nM.

International audience; A potential means to improve the ef®cacy of steric-blocking antisense oligonucleotides (ON) is to increase their af®nity for a target RNA. The grafting of cationic amino groups to the backbone of the ON is one way to achieve this, as it reduces the electro-static repulsion between the ON and its target. We have examined the duplex stabilising effects of introducing cationic phosphoramidate internucle-oside linkages into ON with a non-natural a-anomeric con®guration. Cationic a-ON bound with high af®nity to single-stranded DNA and RNA targets. Duplex stabilisation was proportional to the number of cationic modi®cations, with fully cationic ON having particularly high thermal stability. The average stabilisation was greatly increased at low ionic strength. The duplex formed between cationic a-ON and their RNA targets were not substrates for RNase H. The penalty in T m in¯icted by a single mis-match, however, was high; suggesting that they are well suited as sequence-speci®c, steric-blocking, antisense agents. Using a well-described target sequence in the internal ribosome entry site of the human hepatitis C virus, we have con®rmed this potential in a cell-free translation assay as well as in a whole cell assay. Interestingly, no vectorisation was necessary for the cationic a-ON in cell culture.

Tricyclo (tc)-DNA belongs to the class of conformationally constrained DNA analogs that show enhanced binding properties to DNA and RNA. We prepared tc-oligonucleotides up to 17 nt in length, and evaluated their binding efficiency and selectivity towards complementary RNA, their biological stability in serum, their RNase H inducing potential and their antisense activity in a cellular assay. Relative to RNA or 2'-O-Me-phosphorothioate (PS)-RNA, fully modified tc-oligodeoxynucleotides, 10-17 nt in length, show enhanced selectivity and enhanced thermal stability by approximately 1 degrees C/modification in binding to RNA targets. Tricyclodeoxyoligonucleotides are completely stable in heat-deactivated fetal calf serum at 37 degree C. Moreover, tc-DNA-RNA duplexes are not substrates for RNase H. To test for antisense effects in vivo, we used HeLa cell lines stably expressing the human beta-globin gene with two different point mutations in the second intron. These mutations lead to the inclusion of an aberrant exon in beta-globin mRNA. Lipofectamine-mediated delivery of a 17mer tc-oligodeoxynucleotide complementary to the 3'-cryptic splice site results in correction of aberrant splicing already at nanomolar concentrations with up to 100-fold enhanced efficiency relative to a 2'-O-Me-PS-RNA oligonucleotide of the same length and sequence. In contrast to 2'-O-Me-PS-RNA, tc-DNA shows antisense activity even in the absence of lipofectamine, albeit only at much higher oligonucleotide concentrations.

Hybrids of RNA and arabinonucleic acid (ANA) as well as the 2'-fluoro-ANA analog (2'F-ANA) were recently shown to be substrates of the enzyme RNase H. Although RNase H binds to double-stranded RNA, no cleavage occurs with such duplexes. Therefore, knowledge of the structure of ANA/RNA hybrids may prove helpful in the design of future antisense oligonucleotide analogs. In this study, we have determined the NMR solution structures of ANA/RNA and DNA/RNA hairpin duplexes and compared them to the recently published structure of a 2'F-ANA/RNA hairpin duplex. We demonstrate here that the sugars of RNA nucleotides of the ANA/RNA hairpin stem adopt the C3'-endo (north, A-form) conformation, whereas those of the ANA strand adopt a 'rigid' O4'-endo (east) sugar pucker. The DNA strand of the DNA/RNA hairpin stem is flexible, but the average DNA/RNA hairpin structural parameters are close to the ANA/RNA and 2'F-ANA/RNA hairpin parameters. The minor groove width of ANA/RNA, 2'F-ANA/RNA and DNA/RNA helices is 9.0 +/- 0.5 A, a value that is intermediate between that of A- and B-form duplexes. These results rationalize the ability of ANA/RNA and 2'F-ANA/RNA hybrids to elicit RNase H activity.

Antisense oligonucleotides provide a powerful tool in order to determine the consequences of the reduced expression of a selected target gene and may include target validation and therapeutic applications. Methods of predicting optimum antisense sites are not always effective. We have compared the efficacy of antisense oligonucleotides, which were selected in vitro using random combinatorial oligonucleotide libraries of differing length and complexity, upon putative target sites within TNFalpha mRNA. The relationship of specific target site accessibility and oligonucleotide efficacy with respect to these parameters proved to be complex. Modification of the length of the recognition sequence of the oligonucleotide library illustrated that independent target sites demonstrated a preference for antisense oligonucleotides of a defined and independent optimal length. The efficacy of antisense oligonucleotide sequences selected in vitro paralleled that observed in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. The application of methylphosphonate:phosphodiester chimaeric oligonucleotides to U937 cells reduced mRNA levels to up to 19.8% that of the untreated cell population. This approach provides a predictive means to profile any mRNA of known sequence with respect to the identification and optimisation of sites accessible to antisense oligonucleotide activity.

The internal ribosome entry site (IRES) of hepatitis C virus (HCV) RNA contains >300 bases of highly conserved 5′-terminal sequence, most of it in the uncapped 5′-untranslated region (5′-UTR) upstream from the single AUG initiator triplet at which translation of the HCV polyprotein begins. Although progress has been made in defining singularities like the RNA pseudoknot near this AUG, the sequence and structural features of the HCV IRES which stimulate accurate and efficient initiation of protein synthesis are only partially defined. Here we report that a region further upstream from the AUG, stem–loop II of the HCV IRES, also contains an element of local tertiary structure which we have detected using RNase H cleavage and have mapped using the singular ability of two bases therein to undergo covalent intra-chain crosslinking stimulated by UV light. This pre-existing element maps to two non-contiguous stretches of the HCV IRES sequence, residues 53–68 and 103–117. Several earlier studies have shown that the correct sequence between bases 45 and 70 of the HCV IRES stem–loop II domain is required for initiation of protein synthesis. Because features of local tertiary structure like the one we report here are often associated with protein binding, we propose that the HCV stem–loop II element is directly involved in IRES action.

Accurately identifying accessible sites in RNA is a critical prerequisite for optimising the cleavage efficiency of hammerhead ribozymes and other small nucleozymes. Here we describe a simple RNase H-based procedure to rapidly identify hammerhead ribozyme-accessible sites in gene length RNAs. Twelve semi-randomised RNA–DNA–RNA chimeric oligonucleotide probes, known as ‘gapmers’, were used to direct RNase H cleavage of transcripts with the specificity expected for hammerhead ribozymes, i.e. after NUH sites (where H is A, C or U). Cleavage sites were identified simply by the mobility of RNase H cleavage products relative to RNA markers in denaturing polyacrylamide gels. Sites were identified in transcripts encoding human interleukin-2 and platelet-derived growth factor. Thirteen minimised hammerhead ribozymes, miniribozymes (Mrz), were synthesised and in vitro cleavage efficiency (37°C, pH 7.6 and 1 mM MgCl2) at each site was analysed. Of the 13 Mrz, five were highly effective, demonstrating good initial rate constants and extents of cleavage. The speed and accuracy of this method commends its use in screening for hammerhead-accessible sites.

Three different base paired stems form between U2 and U6 snRNA over the course of the mRNA splicing reaction (helices I, II and III). One possible function of U2/U6 helix II is to facilitate subsequent U2/U6 helix I and III interactions, which participate directly in catalysis. Using an in vitro trans-splicing assay, we investigated the function of sequences located just upstream from the branch site (BS). We find that these upstream sequences are essential for stable binding of U2 to the branch region, and for U2/U6 helix II formation, but not for initial U2/BS pairing. We also show that non-functional upstream sequences cause U2 snRNA stem–loop IIa to be exposed to dimethylsulfate modification, perhaps reflecting a U2 snRNA conformational change and/or loss of SF3b proteins. Our data suggest that initial binding of U2 snRNP to the BS region must be stabilized by an interaction with upstream sequences before U2/U6 helix II can form or U2 stem–loop IIa can participate in spliceosome assembly.

Recently, hybrids of RNA and D-arabinonucleic acids (ANA) as well as the 2'-deoxy-2'-fluoro-D-arabinonucleic acid analog (2'F-ANA) were shown to be substrates of RNase H. This enzyme is believed to be involved in the primary mechanism by which antisense oligonucleotides cause a reduction in target RNA levels in vivo. To gain a better understanding of the properties of arabinose based oligonucleotides, we have prepared a series of 2'F-ANA sequences of homopolymeric (A and T) and mixed base composition (A, T, G and C). UV thermal melting and circular dichroic (CD) studies were used to ascertain the thermodynamic stability and helical conformation of 2'F-ANA/RNA and 2'F-ANA/DNA hybrids. It is shown that 2'F-ANA has enhanced RNA affinity relative to that of DNA and phosphorothioate DNA. The 2'-fluoroarabino modification showed favorable pairing to single-stranded DNA also. This is in sharp contrast to ANA, which forms weak ANA/DNA hybrids at best. According to the measured thermodynamic parameters for duplex formation, the increased stability of hybrids formed by 2'F-ANA (e.g., 2'F-ANA/RNA) appears to originate from conformational pre-organization of the fluorinated sugars and a favorable enthalpy of hybridization. In addition, NMR spectroscopy revealed a five-bond coupling between the 2'F and the base protons (H6/H8) of 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides. This observation is suggestive of a through-space interaction between 2'F and H6/H8 atoms. CD experiments indicate that 2'F-ANA/RNA hybrids adopt an 'A-like' structure and show more resemblance to DNA/RNA hybrids than to the pure RNA/RNA duplex. This feature is believed to be an important factor in the mechanism that allows RNase H to discriminate between 2'F-ANA/RNA (or DNA/RNA) and RNA/RNA duplexes.

Most yeast strains carry a cytoplasmic double-stranded RNA (dsRNA) molecule called W, of 2.5 kb in size. We have cloned and sequenced most of W genome (1), and we proposed that W (+) strands were identical to 20S RNA, a single-stranded RNA (ssRNA) species, whose copy number is highly induced under stress conditions. Recently it was proposed that 20S RNA was circular (2). In this paper, however, we demonstrate that both W dsRNA and 20S RNA are linear. Linearity of W dsRNA is shown by the stoichiometric labelling of both strands of W with 32P-pCp and T4 RNA ligase. The last 3' end nucleotide of both strands is about 70 to 80% C and 20 to 30% A. Linearity of 20S RNA is directly demonstrated by a site-specific cleavage of 20S RNA with RNase H, using an oligodeoxynucleotide complementary to an internal site of 20S RNA. The cleavage produced not one but two RNA fragments expected from the linearity of 20S RNA., This research was supported by Grant B1088-0247 from The Comisión Interministerial de Ciencia y Tecnología (CICYT), Spain. Nieves Rodriguez-Cousiño is the recipient of a fellowship from the University of Salamanca, Spain.