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1.
Nucleic Acids Res ; 49(5): 2721-2739, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33577678

RESUMO

We recently found that toxic PS-ASOs can cause P54nrb and PSF nucleolar mislocalization in an RNase H1-dependent manner. To better understand the underlying mechanisms of these observations, here we utilize different biochemical approaches to demonstrate that PS-ASO binding can alter the conformations of the bound proteins, as illustrated using recombinant RNase H1, P54nrb, PSF proteins and various isolated domains. While, in general, binding of PS-ASOs or ASO/RNA duplexes stabilizes the conformations of these proteins, PS-ASO binding may also cause the unfolding of RNase H1, including both the hybrid binding domain and the catalytic domain. The extent of conformational change correlates with the binding affinity of PS-ASOs to the proteins. Consequently, PS-ASO binding to RNase H1 induces the interaction of RNase H1 with P54nrb or PSF in a 2'-modification and sequence dependent manner, and toxic PS-ASOs tend to induce more interactions than non-toxic PS-ASOs. PS-ASO binding also enhances the interaction between P54nrb and PSF. However, the interaction between RNase H1 and P32 protein can be disrupted upon binding of PS-ASOs. Together, these results suggest that stronger binding of PS-ASOs can cause greater conformational changes of the bound proteins, subsequently affecting protein-protein interactions. These observations thus provide deeper understanding of the molecular basis of PS-ASO-induced protein mislocalization or degradation observed in cells and advance our understanding of why some PS-ASOs are cytotoxic.


Assuntos
Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Fosforotioatos/metabolismo , Ribonuclease H/metabolismo , Linhagem Celular , Quimotripsina , Humanos , Proteínas Nucleares/metabolismo , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Fosforotioatos/química , Ligação Proteica , Conformação Proteica , Sinais Direcionadores de Proteínas , RNA/metabolismo , Ribonuclease H/química
2.
J Vis Exp ; (167)2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33554969

RESUMO

The three-stranded nucleic acid structure, R-loop, is increasingly recognized for its role in gene regulation. Initially, R-loops were thought to be the by-products of transcription; but recent findings of fewer R-loops in diseased cells made it clear that R-loops have functional roles in a variety of human cells. Next, it is critical to understand the roles of R-loops and how cells balance their abundance. A challenge in the field is the quantitation of R-loops since much of the work relies on the S9.6 monoclonal antibody whose specificity for RNA-DNA hybrids has been questioned. Here, we use dot-blots with the S9.6 antibody to quantify R-loops and show the sensitivity and specificity of this assay with RNase H, RNase T1, and RNase III that cleave RNA-DNA hybrids, single-stranded RNA, and double-stranded RNA, respectively. This method is highly reproducible, uses general laboratory equipment and reagents, and provides results within two days. This assay can be used in research and clinical settings to quantify R-loops and assess the effect of mutations in genes such as senataxin on R-loop abundance.


Assuntos
Immunoblotting , Estruturas R-Loop , Anticorpos/metabolismo , DNA/isolamento & purificação , Fibroblastos/metabolismo , Humanos , Ácidos Nucleicos Heteroduplexes/metabolismo , Oligonucleotídeos/metabolismo , Estruturas R-Loop/genética , RNA/genética , Ribonuclease H/metabolismo , Ribonucleases/metabolismo
3.
Nucleic Acids Res ; 48(17): 9840-9858, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32870273

RESUMO

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.


Assuntos
Oligonucleotídeos Antissenso/genética , RNA Mensageiro/genética , Ribonuclease H/metabolismo , Animais , Pareamento de Bases , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oligonucleotídeos Antissenso/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo
4.
Mol Cell ; 79(3): 425-442.e7, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32615088

RESUMO

Double-strand breaks (DSBs) are the most deleterious DNA lesions, which, if left unrepaired, may lead to genome instability or cell death. Here, we report that, in response to DSBs, the RNA methyltransferase METTL3 is activated by ATM-mediated phosphorylation at S43. Phosphorylated METTL3 is then localized to DNA damage sites, where it methylates the N6 position of adenosine (m6A) in DNA damage-associated RNAs, which recruits the m6A reader protein YTHDC1 for protection. In this way, the METTL3-m6A-YTHDC1 axis modulates accumulation of DNA-RNA hybrids at DSBs sites, which then recruit RAD51 and BRCA1 for homologous recombination (HR)-mediated repair. METTL3-deficient cells display defective HR, accumulation of unrepaired DSBs, and genome instability. Accordingly, depletion of METTL3 significantly enhances the sensitivity of cancer cells and murine xenografts to DNA damage-based therapy. These findings uncover the function of METTL3 and YTHDC1 in HR-mediated DSB repair, which may have implications for cancer therapy.


Assuntos
Adenosina/análogos & derivados , Neoplasias de Cabeça e Pescoço/genética , Metiltransferases/genética , Proteínas do Tecido Nervoso/genética , Fatores de Processamento de RNA/genética , Reparo de DNA por Recombinação/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Adenosina/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Bleomicina/farmacologia , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Células HEK293 , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas do Tecido Nervoso/metabolismo , Hibridização de Ácido Nucleico , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Nature ; 585(7824): 298-302, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32669707

RESUMO

Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli1,2. Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as 'red laser'). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease.


Assuntos
Nucléolo Celular/enzimologia , Nucléolo Celular/genética , DNA Ribossômico/genética , RNA Polimerase II/metabolismo , RNA não Traduzido/biossíntese , RNA não Traduzido/genética , Ribossomos/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Nucléolo Celular/fisiologia , DNA Helicases/metabolismo , DNA Intergênico/genética , Humanos , Enzimas Multifuncionais/metabolismo , Biossíntese de Proteínas , Estruturas R-Loop , RNA Helicases/metabolismo , RNA Polimerase I/antagonistas & inibidores , RNA Polimerase I/metabolismo , Ribonuclease H/metabolismo , Ribossomos/química , Ribossomos/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologia
6.
Nucleic Acids Res ; 48(10): 5235-5253, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32356888

RESUMO

Antisense oligonucleotides (ASOs) interact with target RNAs via hybridization to modulate gene expression through different mechanisms. ASO therapeutics are chemically modified and include phosphorothioate (PS) backbone modifications and different ribose and base modifications to improve pharmacological properties. Modified PS ASOs display better binding affinity to the target RNAs and increased binding to proteins. Moreover, PS ASO protein interactions can affect many aspects of their performance, including distribution and tissue delivery, cellular uptake, intracellular trafficking, potency and toxicity. In this review, we summarize recent progress in understanding PS ASO protein interactions, highlighting the proteins with which PS ASOs interact, the influence of PS ASO protein interactions on ASO performance, and the structure activity relationships of PS ASO modification and protein interactions. A detailed understanding of these interactions can aid in the design of safer and more potent ASO drugs, as illustrated by recent findings that altering ASO chemical modifications dramatically improves therapeutic index.


Assuntos
Oligonucleotídeos Fosforotioatos/química , Proteínas/química , Membrana Celular/química , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Ligantes , Oligonucleotídeos Fosforotioatos/metabolismo , Oligonucleotídeos Fosforotioatos/farmacologia , Oligonucleotídeos Fosforotioatos/toxicidade , Ligação Proteica , Domínios Proteicos , Proteínas/metabolismo , Proteínas/toxicidade , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribonuclease H/química , Ribonuclease H/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
7.
Nat Struct Mol Biol ; 27(5): 424-437, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32398827

RESUMO

Oncogene activation during tumorigenesis generates DNA replication stress, a known driver of genome rearrangements. In response to replication stress, certain loci, such as common fragile sites and telomeres, remain under-replicated during interphase and subsequently complete locus duplication in mitosis in a process known as 'MiDAS'. Here, we demonstrate that RTEL1 (regulator of telomere elongation helicase 1) has a genome-wide role in MiDAS at loci prone to form G-quadruplex-associated R-loops, in a process that is dependent on its helicase function. We reveal that SLX4 is required for the timely recruitment of RTEL1 to the affected loci, which in turn facilitates recruitment of other proteins required for MiDAS, including RAD52 and POLD3. Our findings demonstrate that RTEL1 is required for MiDAS and suggest that RTEL1 maintains genome stability by resolving conflicts that can arise between the replication and transcription machineries.


Assuntos
DNA Helicases/genética , DNA Helicases/metabolismo , Quadruplex G , Genoma Humano/genética , Mitose , Animais , Linhagem Celular , DNA Helicases/química , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Instabilidade Genômica , Humanos , Imunoprecipitação , Camundongos , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Conformação de Ácido Nucleico , RNA Helicases/genética , RNA Helicases/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinases/genética , Recombinases/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo
8.
Nat Commun ; 11(1): 2447, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415081

RESUMO

Despite the abundance of ribonucleoside monophosphates (rNMPs) in DNA, sites of rNMP incorporation remain poorly characterized. Here, by using ribose-seq and Ribose-Map techniques, we built and analyzed high-throughput sequencing libraries of rNMPs derived from mitochondrial and nuclear DNA of budding and fission yeast. We reveal both common and unique features of rNMP sites among yeast species and strains, and between wild type and different ribonuclease H-mutant genotypes. We demonstrate that the rNMPs are not randomly incorporated in DNA. We highlight signatures and patterns of rNMPs, including sites within trinucleotide-repeat tracts. Our results uncover that the deoxyribonucleotide immediately upstream of the rNMPs has a strong influence on rNMP distribution, suggesting a mechanism of rNMP accommodation by DNA polymerases as a driving force of rNMP incorporation. Consistently, we find deoxyadenosine upstream from the most abundant genomic rCMPs and rGMPs. This study establishes a framework to better understand mechanisms of rNMP incorporation in DNA.


Assuntos
Citosina/metabolismo , DNA Fúngico/genética , Desoxiadenosinas/metabolismo , Genoma Fúngico , Guanosina/metabolismo , Ribonucleotídeos/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Bases , Núcleo Celular/genética , DNA Mitocondrial/genética , Genoma Mitocondrial , Sequências Repetitivas de Ácido Nucleico/genética , Ribonuclease H/metabolismo , Schizosaccharomyces/genética
9.
PLoS One ; 15(2): e0228774, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32069311

RESUMO

Ribonuclease H2 (RNase H2) exhibits both single ribonucleotide excision activity (activity A) and RNA strand degrading activity (activity B). Val143 of human RNase H2 is located at the active site and is conserved in eukaryotic RNase H2. In this study, we explored the role of Val143 in catalytic activity and substrate specificity. Nineteen single variants at amino acid position 143 were expressed in E. coli, and all variants except for V143C and V143M were purified from the cells. When the activity of the wild-type human RNase H2 (WT) was set as 100%, the relative activities A and B of the 17 variants were in the range of 0.05-130 and 0.02-42%, respectively. When the ratio of the relative activity A to the relative activity B of WT was set as 1, the ratios of the 17 variants were in the range of 0.2-5.7. This indicates that valine is optimal for balancing the two activities. The ratios for V143Y and V143W were relatively high (5.6 and 5.5, respectively), suggesting that the bulky residues like tyrosine and tryptophan at position 143 caused steric hindrance with the 2'-OH of the sugar moiety of the ribonucleotide at the 5' side of the scissile phosphodiester bond. The ratio for V143Q was relatively low (0.2). These results suggested that Val143 is not critical for, but plays a role in determining catalytic activity and substrate specificity.


Assuntos
Biocatálise , Ribonuclease H/química , Ribonuclease H/metabolismo , Valina , Sequência de Aminoácidos , Domínio Catalítico , Humanos , Modelos Moleculares , Mutação , Ribonuclease H/genética , Especificidade por Substrato
10.
Mol Cell ; 77(5): 1032-1043.e4, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31924447

RESUMO

An attractive approach to reduce gene expression is via the use of antisense oligonucleotides (ASOs) that harness the RNase H1 mechanism. Here we show that RNase H ASOs targeted to introns or exons robustly reduce the level of spliced RNA associated with chromatin. Surprisingly, intron-targeted ASOs reduce the level of pre-mRNA associated with chromatin to a greater extent than exon-targeted ASOs. This indicates that exon-targeted ASOs achieve full activity after the pre-mRNA has undergone splicing, but before the mRNA is released from chromatin. Even though RNase H ASOs can reduce the level of RNA associated with chromatin, the effect of ASO-directed RNA degradation on transcription has never been documented. Here we show that intron-targeted ASOs and, to a lesser extent, exon-targeted ASOs cause RNA polymerase II (Pol II) transcription termination in cultured cells and mice. Furthermore, ASO-directed transcription termination is mediated by the nuclear exonuclease XRN2.


Assuntos
Cromatina/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Precursores de RNA/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Ribonuclease H/metabolismo , Terminação da Transcrição Genética , Animais , Cromatina/genética , Éxons , Exorribonucleases/genética , Exorribonucleases/metabolismo , Feminino , Células HCT116 , Humanos , Íntrons , Camundongos Endogâmicos C57BL , Modelos Genéticos , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Oligonucleotídeos Antissenso/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Precursores de RNA/genética , RNA Mensageiro/genética , Ribonuclease H/genética , Fatores de Tempo
11.
Genes Dev ; 34(1-2): 132-145, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31805520

RESUMO

The allosteric and torpedo models have been used for 30 yr to explain how transcription terminates on protein-coding genes. The former invokes termination via conformational changes in the transcription complex and the latter proposes that degradation of the downstream product of poly(A) signal (PAS) processing is important. Here, we describe a single mechanism incorporating features of both models. We show that termination is completely abolished by rapid elimination of CPSF73, which causes very extensive transcriptional readthrough genome-wide. This is because CPSF73 functions upstream of modifications to the elongation complex and provides an entry site for the XRN2 torpedo. Rapid depletion of XRN2 enriches these events that we show are underpinned by protein phosphatase 1 (PP1) activity, the inhibition of which extends readthrough in the absence of XRN2. Our results suggest a combined allosteric/torpedo mechanism, in which PP1-dependent slowing down of polymerases over termination regions facilitates their pursuit/capture by XRN2 following PAS processing.


Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Terminação da Transcrição Genética/fisiologia , Linhagem Celular , Fator de Especificidade de Clivagem e Poliadenilação/genética , Exorribonucleases/metabolismo , Deleção de Genes , Células HCT116 , Humanos , RNA/metabolismo , RNA Polimerase II/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Ribonuclease H/metabolismo
12.
Nucleic Acids Res ; 48(1): 63-74, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31754711

RESUMO

The introduction of non-bridging phosphorothioate (PS) linkages in oligonucleotides has been instrumental for the development of RNA therapeutics and antisense oligonucleotides. This modification offers significantly increased metabolic stability as well as improved pharmacokinetic properties. However, due to the chiral nature of the phosphorothioate, every PS group doubles the amount of possible stereoisomers. Thus PS oligonucleotides are generally obtained as an inseparable mixture of a multitude of diastereoisomeric compounds. Herein, we describe the introduction of non-chiral 3' thiophosphate linkages into antisense oligonucleotides and report their in vitro as well as in vivo activity. The obtained results are carefully investigated for the individual parameters contributing to antisense activity of 3' and 5' thiophosphate modified oligonucleotides (target binding, RNase H recruitment, nuclease stability). We conclude that nuclease stability is the major challenge for this approach. These results highlight the importance of selecting meaningful in vitro experiments particularly when examining hitherto unexplored chemical modifications.


Assuntos
Apolipoproteína B-100/genética , Oligonucleotídeos/genética , Fosfatos/química , Oligonucleotídeos Fosforotioatos/genética , RNA Longo não Codificante/genética , Animais , Apolipoproteína B-100/antagonistas & inibidores , Apolipoproteína B-100/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Rim/citologia , Rim/metabolismo , Fígado/citologia , Fígado/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Oligonucleotídeos/síntese química , Oligonucleotídeos/metabolismo , Fosfatos/metabolismo , Oligonucleotídeos Fosforotioatos/síntese química , Oligonucleotídeos Fosforotioatos/metabolismo , Estabilidade de RNA , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , Ribonuclease H/química , Ribonuclease H/metabolismo , Estereoisomerismo
13.
Chem Commun (Camb) ; 56(4): 639-642, 2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31833487

RESUMO

In this work, we have developed a novel DNAzyme motor initiated by endogenous enzyme for sensitive imaging of intracellular RNase H activity.


Assuntos
DNA Catalítico/metabolismo , Imagem Óptica , Ribonuclease H/metabolismo , Sobrevivência Celular , Células HeLa , Células Hep G2 , Humanos , Células MCF-7 , Microscopia Confocal , Microscopia de Fluorescência
14.
Chemistry ; 26(6): 1368-1379, 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31682037

RESUMO

Off-target effects remain a significant challenge in the therapeutic use of gapmer antisense oligonucleotides (AONs). Over the years various modifications have been synthesized and incorporated into AONs, however, precise control of RNase H-induced cleavage and target sequence selectivity has yet to be realized. Herein, the synthesis of the uracil and cytosine derivatives of a novel class of 2'-deoxy-2'-fluoro-3'-C-hydroxymethyl-ß-d-lyxo-configured nucleotides has been accomplished and the target molecules have been incorporated into AONs. Experiments on exonuclease degradation showed improved nucleolytic stability relative to the unmodified control. Upon the introduction of one or two of the novel 2'-fluoro-3'-C-hydroxymethyl nucleotides as modifications in the gap region of a gapmer AON was associated with efficient RNase H-mediated cleavage of the RNA strand of the corresponding AON:RNA duplex. Notably, a tailored single cleavage event could be engineered depending on the positioning of a single modification. The effect of single mismatched base pairs was scanned along the full gap region demonstrating that the modification enables a remarkable specificity of RNase H cleavage. A cell-based model system was used to demonstrate the potential of gapmer AONs containing the novel modification to mediate gene silencing.


Assuntos
Inativação Gênica , Nucleotídeos/química , Oligonucleotídeos Antissenso/química , Ribonuclease H/metabolismo , Sequência de Bases , Estabilidade Enzimática , Células HeLa , Humanos , Concentração Inibidora 50 , Desnaturação de Ácido Nucleico , Nucleotídeos/metabolismo , Oligonucleotídeos Antissenso/metabolismo , RNA/química , RNA/metabolismo , Temperatura , Transfecção
15.
EMBO J ; 39(3): e102309, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31833079

RESUMO

The duplication of the eukaryotic genome is an intricate process that has to be tightly safe-guarded. One of the most frequently occurring errors during DNA synthesis is the mis-insertion of a ribonucleotide instead of a deoxyribonucleotide. Ribonucleotide excision repair (RER) is initiated by RNase H2 and results in error-free removal of such mis-incorporated ribonucleotides. If left unrepaired, DNA-embedded ribonucleotides result in a variety of alterations within chromosomal DNA, which ultimately lead to genome instability. Here, we review how genomic ribonucleotides lead to chromosomal aberrations and discuss how the tight regulation of RER timing may be important for preventing unwanted DNA damage. We describe the structural impact of unrepaired ribonucleotides on DNA and chromatin and comment on the potential consequences for cellular fitness. In the context of the molecular mechanisms associated with faulty RER, we have placed an emphasis on how and why increased levels of genomic ribonucleotides are associated with severe autoimmune syndromes, neuropathology, and cancer. In addition, we discuss therapeutic directions that could be followed for pathologies associated with defective removal of ribonucleotides from double-stranded DNA.


Assuntos
Cromatina/metabolismo , DNA/metabolismo , Ribonuclease H/metabolismo , Animais , Reparo do DNA , Eucariotos , Aptidão Genética , Instabilidade Genômica , Humanos
17.
DNA Repair (Amst) ; 84: 102736, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31761672

RESUMO

Eukaryotic RNases H2 have dual functions in initiating the removal of ribonucleoside monophosphates (rNMPs) incorporated by DNA polymerases during DNA synthesis and in cleaving the RNA moiety of RNA/DNA hybrids formed during transcription and retrotransposition. The other major cellular RNase H, RNase H1, shares the hybrid processing activity, but not all substrates. After RNase H2 incision at the rNMPs in DNA the Ribonucleotide Excision Repair (RER) pathway completes the removal, restoring dsDNA. The development of the RNase H2-RED (Ribonucleotide Excision Defective) mutant enzyme, which can process RNA/DNA hybrids but is unable to cleave rNMPs embedded in DNA has unlinked the two activities and illuminated the roles of RNase H2 in cellular metabolism. Studies mostly in Saccharomyces cerevisiae, have shown both activities of RNase H2 are necessary to maintain genome integrity and that RNase H1 and H2 have overlapping as well as distinct RNA/DNA hybrid substrates. In mouse RNase H2-RED confirmed that rNMPs in DNA during embryogenesis induce lethality in a p53-dependent DNA damage response. In mammalian cell cultures, RNase H2-RED helped identifying DNA lesions produced by Top1 cleavage at rNMPs and led to determine that RNase H2 participates in the retrotransposition of LINE-1 elements. In this review, we summarize the studies and conclusions reached by utilization of RNase H2-RED enzyme in different model systems.


Assuntos
Reparo do DNA , Ribonuclease H/metabolismo , Animais , Humanos , Ribonuclease H/química , Ribonuclease H/genética , Ribonucleotídeos/genética
18.
Cell Rep ; 29(9): 2890-2900.e5, 2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31775053

RESUMO

RNA-DNA hybrids are tightly regulated to ensure genome integrity. The RNase H enzymes RNase H1 and H2 contribute to chromosomal stability through the removal of RNA-DNA hybrids. Loss of RNase H2 function is implicated in human diseases of the nervous system and cancer. To better understand RNA-DNA hybrid dynamics, we focused on elucidating the regulation of the RNase H enzymes themselves. Using yeast as a model system, we demonstrate that RNase H1 and H2 are controlled in different manners. RNase H2 has strict cell cycle requirements, in that it has an essential function in G2/M for both R-loop processing and ribonucleotide excision repair. RNase H1, however, can function independently of the cell cycle to remove R-loops and appears to become activated in response to high R-loop loads. These results provide us with a more complete understanding of how and when RNA-DNA hybrids are acted upon by the RNase H enzymes.


Assuntos
DNA/metabolismo , RNA/metabolismo , Ribonuclease H/metabolismo , Humanos
19.
Anal Chem ; 91(23): 15138-15146, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31687802

RESUMO

Alternative splicing of mRNA precursors is a key process in gene regulation, contributing to the diversity of proteomes by the alternative selection of exonic sequences. Alterations in this mechanism are associated with most cancers, enhancing their proliferation and survival, and can be employed as cancer biomarkers. Label-free optical biosensors are ideal tools for the highly sensitive and label-free analysis of nucleic acids. However, their application for alternative splicing analysis has been hampered due to the formation of complex and intricate long-range base-pairing interactions which make the direct detection in mRNA isoforms difficult. To solve this bottleneck, we introduce a methodology for the generation of length-controlled RNA fragments from purified total RNA, which can be easily detected by the biosensor. The methodology seizes RNase H enzyme activity to degrade the upstream and downstream RNA segments flanking the target sequence upon hybridization to specific DNA oligos. It allows the fast and direct monitoring of Fas gene alternative splicing in real time, employing a surface plasmon resonance biosensor. We demonstrate the selective and specific detection of mRNA fragments in the pM-nM concentration range, reducing quantification errors and showing 81% accuracy when compared to RT-qPCR. The site-specific cleavage outperformed random RNA hydrolysis by increasing the detection accuracy by 20%, making this methodology particularly appropriate for label-free quantification of alternative splicing events in complex samples.


Assuntos
Processamento Alternativo , Técnicas Biossensoriais/métodos , Processamento de RNA , Técnicas Biossensoriais/normas , Humanos , Reprodutibilidade dos Testes , Ribonuclease H/metabolismo , Ressonância de Plasmônio de Superfície/métodos
20.
Mol Cell ; 76(4): 600-616.e6, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31679819

RESUMO

Widespread antisense long noncoding RNA (lncRNA) overlap with many protein-coding genes in mammals and emanate from gene promoter, enhancer, and termination regions. However, their origin and biological purpose remain unclear. We show that these antisense lncRNA can be generated by R-loops that form when nascent transcript invades the DNA duplex behind elongating RNA polymerase II (Pol II). Biochemically, R-loops act as intrinsic Pol II promoters to induce de novo RNA synthesis. Furthermore, their removal across the human genome by RNase H1 overexpression causes the selective reduction of antisense transcription. Consequently, we predict that R-loops act to facilitate the synthesis of many gene proximal antisense lncRNA. Not only are R-loops widely associated with DNA damage and repair, but we now show that they have the capacity to promote de novo transcript synthesis that may have aided the evolution of gene regulation.


Assuntos
Genoma Humano , Regiões Promotoras Genéticas , Estruturas R-Loop , RNA Antissenso/biossíntese , RNA Longo não Codificante/biossíntese , Transcrição Genética , Ativação Transcricional , Células HEK293 , Células HeLa , Humanos , RNA Antissenso/genética , RNA Longo não Codificante/genética , Ribonuclease H/metabolismo , Relação Estrutura-Atividade
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