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2.
Nucleic Acids Res ; 49(15): 8777-8784, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34365509

RESUMO

Transcribing RNA polymerase (RNAP) can fall into backtracking, phenomenon when the 3' end of the transcript disengages from the template DNA. Backtracking is caused by sequences of the nucleic acids or by misincorporation of erroneous nucleotides. To resume productive elongation backtracked complexes have to be resolved through hydrolysis of RNA. There is currently no consensus on the mechanism of catalysis of this reaction by Escherichia coli RNAP. Here we used Salinamide A, that we found inhibits RNAP catalytic domain Trigger Loop (TL), to show that the TL is required for RNA cleavage during proofreading of misincorporation events but plays little role during cleavage in sequence-dependent backtracked complexes. Results reveal that backtracking caused by misincorporation is distinct from sequence-dependent backtracking, resulting in different conformations of the 3' end of RNA within the active center. We show that the TL is required to transfer the 3' end of misincorporated transcript from cleavage-inefficient 'misincorporation site' into the cleavage-efficient 'backtracked site', where hydrolysis takes place via transcript-assisted catalysis and is largely independent of the TL. These findings resolve the controversy surrounding mechanism of RNA hydrolysis by E. coli RNA polymerase and indicate that the TL role in RNA cleavage has diverged among bacteria.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , RNA Mensageiro/metabolismo , Elongação da Transcrição Genética , Domínio Catalítico , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/química , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Hidrólise , Clivagem do RNA
3.
BMC Bioinformatics ; 22(1): 380, 2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294042

RESUMO

BACKGROUND: RNA degradation is important for the regulation of gene expression. Despite the identification of proteins and sequences related to deadenylation-dependent RNA degradation in plants, endonucleolytic cleavage-dependent RNA degradation has not been studied in detail. Here, we developed truncated RNA end sequencing in Arabidopsis thaliana to identify cleavage sites and evaluate the efficiency of cleavage at each site. Although several features are related to RNA cleavage efficiency, the effect of each feature on cleavage efficiency has not been evaluated by considering multiple putative determinants in A. thaliana. RESULTS: Cleavage site information was acquired from a previous study, and cleavage efficiency at the site level (CSsite value), which indicates the number of reads at each cleavage site normalized to RNA abundance, was calculated. To identify features related to cleavage efficiency at the site level, multiple putative determinants (features) were used to perform feature selection using the Least Absolute Shrinkage and Selection Operator (LASSO) regression model. The results indicated that whole RNA features were important for the CSsite value, in addition to features around cleavage sites. Whole RNA features related to the translation process and nucleotide frequency around cleavage sites were major determinants of cleavage efficiency. The results were verified in a model constructed using only sequence features, which showed that the prediction accuracy was similar to that determined using all features including the translation process, suggesting that cleavage efficiency can be predicted using only sequence information. The LASSO regression model was validated in exogenous genes, which showed that the model constructed using only sequence information can predict cleavage efficiency in both endogenous and exogenous genes. CONCLUSIONS: Feature selection using the LASSO regression model in A. thaliana identified 155 features. Correlation coefficients revealed that whole RNA features are important for determining cleavage efficiency in addition to features around the cleavage sites. The LASSO regression model can predict cleavage efficiency in endogenous and exogenous genes using only sequence information. The model revealed the significance of the effect of multiple determinants on cleavage efficiency, suggesting that sequence features are important for RNA degradation mechanisms in A. thaliana.


Assuntos
Arabidopsis , Arabidopsis/genética , Proteínas , Clivagem do RNA , Estabilidade de RNA , Análise de Sequência de RNA
4.
Drug Discov Today ; 26(8): 2025-2035, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34147688

RESUMO

The limitations of conventional diagnostic procedures, such as real-time PCR-based methods and serological tests, have led the scientific community to innovate alternative nucleic acid detection approaches for SARS-CoV-2 RNA, thereby addressing the dire need for increased testing. Such approaches aim to provide rapid, accurate, cost-effective, sensitive, and high-throughput detection of SARS-CoV-2 RNA, on multiple specimen types, and without specialized equipment and expertise. The CRISPR-Cas13 system functions as a sequence-specific RNA-sensing tool that has recently been harnessed to develop simplified and flexible testing formats. This review recapitulates technical advances in the most recent CRISPR-Cas13-based methods for SARS-CoV-2/COVID-19 diagnosis. The challenges and opportunities for implementing mass testing using these novel CRISPR-Cas13 platforms are critically analyzed.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19 , Sistemas CRISPR-Cas/fisiologia , RNA Viral/isolamento & purificação , SARS-CoV-2/isolamento & purificação , COVID-19/diagnóstico , COVID-19/virologia , Proteínas Associadas a CRISPR , Humanos , Testes Imediatos/tendências , Clivagem do RNA
5.
Nucleic Acids Res ; 49(12): 6971-6981, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34133739

RESUMO

Artificial evolution experiments typically use libraries of ∼1015 sequences and require multiple rounds of selection to identify rare variants with a desired activity. Based on the simple structures of some aptamers and nucleic acid enzymes, we hypothesized that functional motifs could be isolated from significantly smaller libraries in a single round of selection followed by high-throughput sequencing. To test this idea, we investigated the catalytic potential of DNA architectures in which twelve or fifteen randomized positions were embedded in a scaffold present in all library members. After incubating in either the presence or absence of lead (which promotes the nonenzymatic cleavage of RNA), library members that cleaved themselves at an RNA linkage were purified by PAGE and characterized by high-throughput sequencing. These selections yielded deoxyribozymes with activities 8- to 30-fold lower than those previously isolated under similar conditions from libraries containing 1014 different sequences, indicating that the disadvantage of using a less diverse pool can be surprisingly small. It was also possible to elucidate the sequence requirements and secondary structures of deoxyribozymes without performing additional experiments. Due to its relative simplicity, we anticipate that this approach will accelerate the discovery of new catalytic DNA and RNA motifs.


Assuntos
DNA Catalítico/química , DNA Catalítico/metabolismo , DNA Catalítico/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Clivagem do RNA , Especificidade por Substrato
6.
Int J Mol Sci ; 22(6)2021 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-33799331

RESUMO

Transfer RNA (tRNA) molecules contain various post-transcriptional modifications that are crucial for tRNA stability, translation efficiency, and fidelity. Besides their canonical roles in translation, tRNAs also originate tRNA-derived small RNAs (tsRNAs), a class of small non-coding RNAs with regulatory functions ranging from translation regulation to gene expression control and cellular stress response. Recent evidence indicates that tsRNAs are also modified, however, the impact of tRNA epitranscriptome deregulation on tsRNAs generation is only now beginning to be uncovered. The 5-methyluridine (m5U) modification at position 54 of cytosolic tRNAs is one of the most common and conserved tRNA modifications among species. The tRNA methyltransferase TRMT2A catalyzes this modification, but its biological role remains mostly unexplored. Here, we show that TRMT2A knockdown in human cells induces m5U54 tRNA hypomodification and tsRNA formation. More specifically, m5U54 hypomodification is followed by overexpression of the ribonuclease angiogenin (ANG) that cleaves tRNAs near the anticodon, resulting in accumulation of 5'tRNA-derived stress-induced RNAs (5'tiRNAs), namely 5'tiRNA-GlyGCC and 5'tiRNA-GluCTC, among others. Additionally, transcriptomic analysis confirms that down-regulation of TRMT2A and consequently m5U54 hypomodification impacts the cellular stress response and RNA stability, which is often correlated with tiRNA generation. Accordingly, exposure to oxidative stress conditions induces TRMT2A down-regulation and tiRNA formation in mammalian cells. These results establish a link between tRNA hypomethylation and ANG-dependent tsRNAs formation and unravel m5U54 as a tRNA cleavage protective mark.


Assuntos
Estresse Oxidativo/genética , RNA de Transferência/genética , Ribonuclease Pancreático/genética , tRNA Metiltransferases/genética , Humanos , Clivagem do RNA/genética , Processamento Pós-Transcricional do RNA/genética , Estabilidade de RNA/genética , Pequeno RNA não Traduzido/genética , RNA de Transferência/química , Estresse Fisiológico/genética , Uridina/análogos & derivados , Uridina/genética
7.
Molecules ; 26(6)2021 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-33808835

RESUMO

RNA-targeting therapeutics require highly efficient sequence-specific devices capable of RNA irreversible degradation in vivo. The most developed methods of sequence-specific RNA cleavage, such as siRNA or antisense oligonucleotides (ASO), are currently based on recruitment of either intracellular multi-protein complexes or enzymes, leaving alternative approaches (e.g., ribozymes and DNAzymes) far behind. Recently, site-selective artificial ribonucleases combining the oligonucleotide recognition motifs (or their structural analogues) and catalytically active groups in a single molecular scaffold have been proven to be a great competitor to siRNA and ASO. Using the most efficient catalytic groups, utilising both metal ion-dependent (Cu(II)-2,9-dimethylphenanthroline) and metal ion-free (Tris(2-aminobenzimidazole)) on the one hand and PNA as an RNA recognising oligonucleotide on the other, allowed site-selective artificial RNases to be created with half-lives of 0.5-1 h. Artificial RNases based on the catalytic peptide [(ArgLeu)2Gly]2 were able to take progress a step further by demonstrating an ability to cleave miRNA-21 in tumour cells and provide a significant reduction of tumour growth in mice.


Assuntos
Sequência de Bases , DNA Catalítico/química , Oligonucleotídeos/química , Clivagem do RNA , RNA/química , Ribonucleases/química
8.
BMC Bioinformatics ; 22(1): 63, 2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33568063

RESUMO

BACKGROUND: Human dicer is an enzyme that cleaves pre-miRNAs into miRNAs. Several models have been developed to predict human dicer cleavage sites, including PHDCleav and LBSizeCleav. Given an input sequence, these models can predict whether the sequence contains a cleavage site. However, these models only consider each sequence independently and lack interpretability. Therefore, it is necessary to develop an accurate and explainable predictor, which employs relations between different sequences, to enhance the understanding of the mechanism by which human dicer cleaves pre-miRNA. RESULTS: In this study, we develop an accurate and explainable predictor for human dicer cleavage site - ReCGBM. We design relational features and class features as inputs to a lightGBM model. Computational experiments show that ReCGBM achieves the best performance compared to the existing methods. Further, we find that features in close proximity to the center of pre-miRNA are more important and make a significant contribution to the performance improvement of the developed method. CONCLUSIONS: The results of this study show that ReCGBM is an interpretable and accurate predictor. Besides, the analyses of feature importance show that it might be of particular interest to consider more informative features close to the center of the pre-miRNA in future predictors.


Assuntos
RNA Helicases DEAD-box , MicroRNAs , Precursores de RNA , Ribonuclease III , RNA Helicases DEAD-box/genética , Humanos , MicroRNAs/genética , Clivagem do RNA , Ribonuclease III/genética
9.
Vet Res ; 52(1): 20, 2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33579382

RESUMO

Processing and packaging of herpesvirus genomic DNA is regulated by a packaging-associated terminase complex comprising of viral proteins pUL15, pUL28 and pUL33. Marek's disease virus (MDV) homologs UL28 and UL33 showed conserved functional features with high sequence identity with the corresponding Herpes simplex virus 1 (HSV-1) homologs. As part of the investigations into the role of the UL28 and UL33 homologs of oncogenic MDV for DNA packaging and replication in cultured cells, we generated MDV mutant clones deficient in UL28 or UL33 of full-length MDV genomes. Transfection of UL28- or UL33-deleted BAC DNA into chicken embryo fibroblast (CEF) did not result either in the production of visible virus plaques, or detectable single cell infection after passaging onto fresh CEF cells. However, typical MDV plaques were detectable in CEF transfected with the DNA of revertant mutants where the deleted genes were precisely reinserted. Moreover, the replication defect of the UL28-deficient mutant was completely restored when fragment encoding the full UL28 gene was co-transfected into CEF cells. Viruses recovered from the revertant construct, as well as by the UL28 co-transfection, showed replication ability comparable with parental virus. Furthermore, the transmission electron microscopy study indicated that immature capsids were assembled without the UL28 expression, but with the loss of infectivity. Importantly, predicted three-dimensional structures of UL28 between MDV and HSV-1 suggests conserved function in virus replication. For the first time, these results revealed that both UL28 and UL33 are essential for MDV replication through regulating DNA cleavage and packaging.


Assuntos
DNA Viral/química , Endodesoxirribonucleases/genética , Mardivirus/fisiologia , Receptores de Quimiocinas/genética , Proteínas Virais/genética , Replicação Viral , Sequência de Aminoácidos , Animais , Embrião de Galinha , Endodesoxirribonucleases/química , Endodesoxirribonucleases/metabolismo , Mardivirus/enzimologia , Mardivirus/genética , Clivagem do RNA , Receptores de Quimiocinas/química , Receptores de Quimiocinas/metabolismo , Alinhamento de Sequência , Organismos Livres de Patógenos Específicos , Proteínas Virais/química , Proteínas Virais/metabolismo
10.
Mol Cell ; 81(5): 998-1012.e7, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33440169

RESUMO

Pre-mRNA processing steps are tightly coordinated with transcription in many organisms. To determine how co-transcriptional splicing is integrated with transcription elongation and 3' end formation in mammalian cells, we performed long-read sequencing of individual nascent RNAs and precision run-on sequencing (PRO-seq) during mouse erythropoiesis. Splicing was not accompanied by transcriptional pausing and was detected when RNA polymerase II (Pol II) was within 75-300 nucleotides of 3' splice sites (3'SSs), often during transcription of the downstream exon. Interestingly, several hundred introns displayed abundant splicing intermediates, suggesting that splicing delays can take place between the two catalytic steps. Overall, splicing efficiencies were correlated among introns within the same transcript, and intron retention was associated with inefficient 3' end cleavage. Remarkably, a thalassemia patient-derived mutation introducing a cryptic 3'SS improved both splicing and 3' end cleavage of individual ß-globin transcripts, demonstrating functional coupling between the two co-transcriptional processes as a determinant of productive gene output.


Assuntos
Células Eritroides/metabolismo , Eritropoese/genética , RNA Polimerase II/genética , Splicing de RNA , Elongação da Transcrição Genética , Globinas beta/genética , Animais , Sequência de Bases , Diferenciação Celular , Linhagem Celular Tumoral , Células Eritroides/citologia , Éxons , Humanos , Íntrons , Leucócitos/citologia , Leucócitos/metabolismo , Camundongos , Mutação , Clivagem do RNA , RNA Polimerase II/metabolismo , Sítios de Splice de RNA , Spliceossomos/genética , Spliceossomos/metabolismo , Globinas beta/deficiência , Talassemia beta/genética , Talassemia beta/metabolismo , Talassemia beta/patologia
11.
Mol Cell ; 81(5): 1100-1115.e5, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33472057

RESUMO

Bacteria and archaea apply CRISPR-Cas surveillance complexes to defend against foreign invaders. These invading genetic elements are captured and integrated into the CRISPR array as spacer elements, guiding sequence-specific DNA/RNA targeting and cleavage. Recently, in vivo studies have shown that target RNAs with extended complementarity with repeat sequences flanking the target element (tag:anti-tag pairing) can dramatically reduce RNA cleavage by the type VI-A Cas13a system. Here, we report the cryo-EM structure of Leptotrichia shahii LshCas13acrRNA in complex with target RNA harboring tag:anti-tag pairing complementarity, with the observed conformational changes providing a molecular explanation for inactivation of the composite HEPN domain cleavage activity. These structural insights, together with in vitro biochemical and in vivo cell-based assays on key mutants, define the molecular principles underlying Cas13a's capacity to target and discriminate between self and non-self RNA targets. Our studies illuminate approaches to regulate Cas13a's cleavage activity, thereby influencing Cas13a-mediated biotechnological applications.


Assuntos
Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Endodesoxirribonucleases/química , Leptotrichia/genética , RNA Guia/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Clonagem Molecular , Microscopia Crioeletrônica , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Leptotrichia/metabolismo , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Clivagem do RNA , RNA Guia/genética , RNA Guia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
12.
Nucleic Acids Res ; 49(7): e41, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33511416

RESUMO

Modifications in RNA are numerous (∼170) and in higher numbers compared to DNA (∼5) making the ability to sequence an RNA molecule to identify these modifications highly tenuous using next generation sequencing (NGS). The ability to immobilize an exoribonuclease enzyme, such as XRN1, to a solid support while maintaining its activity and capability to cleave both the canonical and modified ribonucleotides from an intact RNA molecule can be a viable approach for single-molecule RNA sequencing. In this study, we report an enzymatic reactor consisting of covalently attached XRN1 to a solid support as the groundwork for a novel RNA exosequencing technique. The covalent attachment of XRN1 to a plastic solid support was achieved using EDC/NHS coupling chemistry. Studies showed that the solid-phase digestion efficiency of model RNAs was 87.6 ± 2.8%, while the XRN1 solution-phase digestion for the same model was 78.3 ± 4.4%. The ability of immobilized XRN1 to digest methylated RNA containing m6A and m5C ribonucleotides was also demonstrated. The processivity and clipping rate of immobilized XRN1 secured using single-molecule fluorescence measurements of a single RNA transcript demonstrated a clipping rate of 26 ± 5 nt s-1 and a processivity of >10.5 kb at 25°C.


Assuntos
Distrofina/genética , Enzimas Imobilizadas/metabolismo , Exorribonucleases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , RNA/metabolismo , Análise de Sequência de RNA/métodos , Humanos , Clivagem do RNA
13.
Nucleic Acids Res ; 49(4): 2192-2212, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33450025

RESUMO

In Staphylococcus aureus, de novo methionine biosynthesis is regulated by a unique hierarchical pathway involving stringent-response controlled CodY repression in combination with a T-box riboswitch and RNA decay. The T-box riboswitch residing in the 5' untranslated region (met leader RNA) of the S. aureus metICFE-mdh operon controls downstream gene transcription upon interaction with uncharged methionyl-tRNA. met leader and metICFE-mdh (m)RNAs undergo RNase-mediated degradation in a process whose molecular details are poorly understood. Here we determined the secondary structure of the met leader RNA and found the element to harbor, beyond other conserved T-box riboswitch structural features, a terminator helix which is target for RNase III endoribonucleolytic cleavage. As the terminator is a thermodynamically highly stable structure, it also forms posttranscriptionally in met leader/ metICFE-mdh read-through transcripts. Cleavage by RNase III releases the met leader from metICFE-mdh mRNA and initiates RNase J-mediated degradation of the mRNA from the 5'-end. Of note, metICFE-mdh mRNA stability varies over the length of the transcript with a longer lifespan towards the 3'-end. The obtained data suggest that coordinated RNA decay represents another checkpoint in a complex regulatory network that adjusts costly methionine biosynthesis to current metabolic requirements.


Assuntos
Metionina/biossíntese , Óperon , Estabilidade de RNA , RNA Mensageiro/metabolismo , Ribonuclease III/metabolismo , Riboswitch , Staphylococcus aureus/genética , Regiões 5' não Traduzidas , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Conformação de Ácido Nucleico , Clivagem do RNA , Staphylococcus aureus/enzimologia , Staphylococcus aureus/metabolismo
14.
Angew Chem Int Ed Engl ; 60(9): 4782-4788, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33188548

RESUMO

Legionella pneumophila is a deadly bacterial pathogen that has caused numerous Legionnaires' disease outbreaks, where cooling towers were the most common source of exposure. Bacterial culturing is used for L. pneumophila detection, but this method takes approximately 10 days to complete. In this work, an RNA-cleaving fluorogenic DNAzyme, named LP1, was isolated. Extensive characterization revealed that LP1 is reactive with multiple infectious isolates of L. pneumophila but inactive with 25 other common bacterial species. LP1 is likely activated by a protein target, capable of generating a detectable signal in the presence of as few as 10 colony-forming units of L. pneumophila, and able to maintain its activity in cooling tower water from diverse sources. Given that similar DNAzymes have been incorporated into many sensitive assays for bacterial detection, LP1 holds the potential for the development of biosensors for monitoring the contamination of L. pneumophila in exposure sources.


Assuntos
DNA Catalítico/metabolismo , Legionella pneumophila/genética , RNA/metabolismo , Técnicas Biossensoriais , DNA Catalítico/química , DNA Catalítico/isolamento & purificação , Cinética , Conformação de Ácido Nucleico , Clivagem do RNA , Microbiologia da Água
15.
Methods Mol Biol ; 2167: 79-89, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32712916

RESUMO

Deoxyribozymes capable of catalyzing sequence-specific RNA cleavage have broad applications in biotechnology. In vitro selected RNA-cleaving deoxyribozymes normally contain two substrate-binding arms and a central catalytic core region. Here, we describe the systematic characterization and optimization of an RNA-cleaving deoxyribozyme with an unusually short left binding arm, and its special sequence requirement for its optimal catalytic activity.


Assuntos
DNA Catalítico/química , DNA Catalítico/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Ensaios Enzimáticos/métodos , Conformação de Ácido Nucleico , Clivagem do RNA/efeitos dos fármacos , Sequência de Bases , Catálise , Domínio Catalítico , DNA Catalítico/genética , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Íons/química , Cinética , Metais/química , Modelos Moleculares , Oligonucleotídeos/química , Oligonucleotídeos/genética , Clivagem do RNA/genética , Especificidade por Substrato
16.
Cell Rep ; 33(13): 108569, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33378676

RESUMO

The present study demonstrates that topoisomerase 3B (TOP3B) forms both RNA and DNA cleavage complexes (TOP3Bccs) in vivo and reveals a pathway for repairing TOP3Bccs. For inducing and detecting cellular TOP3Bccs, we engineer a "self-trapping" mutant of TOP3B (R338W-TOP3B). Transfection with R338W-TOP3B induces R-loops, genomic damage, and growth defect, which highlights the importance of TOP3Bcc repair mechanisms. To determine how cells repair TOP3Bccs, we deplete tyrosyl-DNA phosphodiesterases (TDP1 and TDP2). TDP2-deficient cells show elevated TOP3Bccs both in DNA and RNA. Conversely, overexpression of TDP2 lowers cellular TOP3Bccs. Using recombinant human TDP2, we demonstrate that TDP2 can process both denatured and proteolyzed TOP3Bccs. We also show that cellular TOP3Bccs are ubiquitinated by the E3 ligase TRIM41 before undergoing proteasomal processing and excision by TDP2.


Assuntos
Reparo do DNA , DNA Topoisomerases Tipo I/fisiologia , Proteínas de Ligação a DNA/fisiologia , DNA/metabolismo , Diester Fosfórico Hidrolases/fisiologia , RNA/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Substituição de Aminoácidos , Clivagem do DNA , Técnicas de Inativação de Genes , Células HCT116 , Células HEK293 , Humanos , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Proteólise , Estruturas R-Loop , Clivagem do RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitinação
17.
Nat Commun ; 11(1): 6031, 2020 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-33247135

RESUMO

Plant spermatogenesis is a complex process that directly affects crop breeding. A rapid change in gene abundance occurs at early meiosis prophase, when gene regulation is selective. However, how these genes are regulated remains unknown. Here, we show that rice reproductive phasiRNAs are essential for the elimination of a specific set of RNAs during meiotic prophase I. These phasiRNAs cleave target mRNAs in a regulatory manner such that one phasiRNA can target more than one gene, and/or a single gene can be targeted by more than one phasiRNA to efficiently silence target genes. Our investigation of phasiRNA-knockdown and PHAS-edited transgenic plants demonstrates that phasiRNAs and their nucleotide variations are required for meiosis progression and fertility. This study highlights the importance of reproductive phasiRNAs for the reprogramming of gene expression during meiotic progression and establishes a basis for future studies on the roles of phasiRNAs with a goal of crop improvement.


Assuntos
Regulação da Expressão Gênica de Plantas , Meiose/genética , Oryza/citologia , Oryza/genética , RNA de Plantas/metabolismo , Sequência de Bases , Fertilidade/genética , Gametogênese Vegetal/genética , Modelos Biológicos , Nucleotídeos/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Pólen/citologia , Pólen/genética , Clivagem do RNA , RNA de Plantas/genética , Reprodutibilidade dos Testes
18.
J Phys Chem Lett ; 11(21): 9298-9303, 2020 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-33085887

RESUMO

The discovery of RNA enzymes, ribozymes, provided strong support to the RNA world hypothesis suggesting that early life evolved from RNAs able to both store genetic information and catalyze biochemical reactions. Moreover, evidence is accumulating that primitive life might have emerged in deep-sea environments and, thus, at high hydrostatic pressures. If true, ribozymes should be able to function under those pressures. In this work, we ask if and possibly how ribozymes could function at high pressures. To this end, we specifically focus on the chemical reaction steps of the self-cleavage catalysis of hairpin ribozyme by employing extensive QM/MM metadynamics simulations. We find that the reaction scenario at high pressures is vastly different than that at ambient conditions, yet the rate-limiting reaction barrier and, thus, the reaction rate are only marginally affected. Therefore, the results indeed suggest that ribozymes would function at high pressures but by following a vastly different reaction scenario.


Assuntos
RNA Catalítico/química , Sequência de Bases , Catálise , Pressão Hidrostática , Cinética , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Clivagem do RNA
19.
Science ; 369(6510): 1470-1476, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32943521

RESUMO

Production of small ribosomal subunits initially requires the formation of a 90S precursor followed by an enigmatic process of restructuring into the primordial pre-40S subunit. We elucidate this process by biochemical and cryo-electron microscopy analysis of intermediates along this pathway in yeast. First, the remodeling RNA helicase Dhr1 engages the 90S pre-ribosome, followed by Utp24 endonuclease-driven RNA cleavage at site A1, thereby separating the 5'-external transcribed spacer (ETS) from 18S ribosomal RNA. Next, the 5'-ETS and 90S assembly factors become dislodged, but this occurs sequentially, not en bloc. Eventually, the primordial pre-40S emerges, still retaining some 90S factors including Dhr1, now ready to unwind the final small nucleolar U3-18S RNA hybrid. Our data shed light on the elusive 90S to pre-40S transition and clarify the principles of assembly and remodeling of large ribonucleoproteins.


Assuntos
RNA Helicases DEAD-box/metabolismo , Proteínas Nucleares/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Microscopia Crioeletrônica , RNA Helicases DEAD-box/química , Proteínas Nucleares/química , Conformação Proteica , Clivagem do RNA , RNA Ribossômico 18S/química , RNA Ribossômico 18S/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/química , Proteínas de Saccharomyces cerevisiae/química
20.
Biochem Biophys Res Commun ; 530(1): 196-202, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32828285

RESUMO

It is well established that U1 snRNP inhibits the cleavage of cryptic polyadenylation site (PAS) within introns, thereby facilitating full-length mRNA transcription for numerous genes in vertebrate cells, yet the underlying mechanism remains poorly understood. Here, by using a model PAS of wdr26 mRNA, we show that U1 snRNP predominantly interferes with the association of PAS with a core 3' processing factor CstF64, which can promote the cleavage step of mRNA 3' processing. Furthermore, we provide evidence that U1A, a component of U1 snRNP, might directly interfere with CstF64 binding on PAS through its RNA binding capacity. Consistently, U1A could potentially associate with U1-suppressed intronic PASs at the transcriptome level in human cells, showing a binding peak ∼50 nt downstream of the cleavage site, as revealed by U1A iCLIP-seq (individual-nucleotide resolution UV crosslinking and immunoprecipitation coupled with RNA sequencing) analysis. Together, our data suggest a molecular mechanism underlying U1 snRNP inhibition of the cleavage step of mRNA 3' processing. More generally, we argue that U1 snRNP might inhibit the usage of cryptic PASs through disturbing the recruitment of core 3' processing factors.


Assuntos
Processamento de Terminações 3' de RNA , RNA Mensageiro/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Células HeLa , Humanos , Poliadenilação , Clivagem do RNA , RNA Mensageiro/genética
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