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1.
Nat Struct Mol Biol ; 28(9): 713-723, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34489609

RESUMO

Human mitochondrial transcripts contain messenger and ribosomal RNAs flanked by transfer RNAs (tRNAs), which are excised by mitochondrial RNase (mtRNase) P and Z to liberate all RNA species. In contrast to nuclear or bacterial RNase P, mtRNase P is not a ribozyme but comprises three protein subunits that carry out RNA cleavage and methylation by unknown mechanisms. Here, we present the cryo-EM structure of human mtRNase P bound to precursor tRNA, which reveals a unique mechanism of substrate recognition and processing. Subunits TRMT10C and SDR5C1 form a subcomplex that binds conserved mitochondrial tRNA elements, including the anticodon loop, and positions the tRNA for methylation. The endonuclease PRORP is recruited and activated through interactions with its PPR and nuclease domains to ensure precise pre-tRNA cleavage. The structure provides the molecular basis for the first step of RNA processing in human mitochondria.


Assuntos
3-Hidroxiacil-CoA Desidrogenases/química , Metiltransferases/química , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , Ribonuclease P/química , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Anticódon/química , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Microscopia Crioeletrônica , Humanos , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Mitocôndrias/enzimologia , Modelos Moleculares , Mutação de Sentido Incorreto , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , RNA Fúngico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ribonuclease P/metabolismo , Especificidade da Espécie , Relação Estrutura-Atividade , Especificidade por Substrato
2.
RNA ; 27(10): 1140-1147, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34253686

RESUMO

Human metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a nuclear long noncoding RNA (lncRNA) that is highly overexpressed in many cancer tissues and plays important roles in tumor progression and metastasis. The MALAT1 primary transcript contains evolutionarily conserved structural elements in its 3'-terminal region: a triple helix forming element called element for nuclear expression (ENE) and a downstream tRNA-like structure called mascRNA. Instead of being polyadenylated, mature MALAT1 is generated by recognition and processing of the mascRNA by RNase P. A genomically encoded A-rich tract at the new 3' end of MALAT1, which is generated upon RNase P cleavage, forms a triple helical structure with the upstream ENE. Triplex formation is vital for stabilization of the mature transcript and for subsequent accumulation and oncogenic activity of MALAT1. Here, we demonstrate that efficient 3'-end maturation of MALAT1 is dependent on an interaction between the A-rich tract and the mascRNA 3' trailer. Using mutational analyses of cell-based reporter accumulation, we show that an extended mascRNA acceptor stem and formation of a single bulged A 5' to the RNase P cleavage site are required for efficient maturation of the nascent MALAT1 3' end. Our results should benefit the development of therapeutic approaches to cancer through targeting MALAT1.


Assuntos
Adenocarcinoma de Pulmão/genética , Neoplasias Pulmonares/genética , Processamento de Terminações 3' de RNA , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Pareamento de Bases , Sequência de Bases , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Ribonuclease P/genética , Ribonuclease P/metabolismo
3.
RNA ; 27(10): 1204-1219, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34266994

RESUMO

In most bacterial type A RNase P RNAs (P RNAs), two major loop-helix tertiary contacts (L8-P4 and L18-P8) help to orient the two independently folding S- and C-domains for concerted recognition of precursor tRNA substrates. Here, we analyze the effects of mutations in these tertiary contacts in P RNAs from three different species: (i) the psychrophilic bacterium Pseudoalteromonas translucida (Ptr), (ii) the mesophilic radiation-resistant bacterium Deinococcus radiodurans (Dra), and (iii) the thermophilic bacterium Thermus thermophilus (Tth). We show by UV melting experiments that simultaneous disruption of these two interdomain contacts has a stabilizing effect on all three P RNAs. This can be inferred from reduced RNA unfolding at lower temperatures and a more concerted unfolding at higher temperatures. Thus, when the two domains tightly interact via the tertiary contacts, one domain facilitates structural transitions in the other. P RNA mutants with disrupted interdomain contacts showed severe kinetic defects that were most pronounced upon simultaneous disruption of the L8-P4 and L18-P8 contacts. At 37°C, the mildest effects were observed for the thermostable Tth RNA. A third interdomain contact, L9-P1, makes only a minor contribution to P RNA tertiary folding. Furthermore, D. radiodurans RNase P RNA forms an additional pseudoknot structure between the P9 and P12 of its S-domain. This interaction was found to be particularly crucial for RNase P holoenzyme activity at near-physiological Mg2+ concentrations (2 mM). We further analyzed an exceptionally stable folding trap of the G,C-rich Tth P RNA.


Assuntos
Deinococcus/genética , Pseudoalteromonas/genética , RNA Bacteriano/genética , RNA de Transferência/genética , Ribonuclease P/genética , Thermus thermophilus/genética , Pareamento de Bases , Sequência de Bases , Deinococcus/metabolismo , Regulação Bacteriana da Expressão Gênica , Cinética , Mutação , Pseudoalteromonas/metabolismo , Processamento de Terminações 3' de RNA , Dobramento de RNA , Estabilidade de RNA , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribonuclease P/metabolismo , Temperatura , Termodinâmica , Thermus thermophilus/metabolismo
4.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199774

RESUMO

Over a thousand nucleus-encoded mitochondrial proteins are imported from the cytoplasm; however, mitochondrial (mt) DNA encodes for a small number of critical proteins and the entire suite of mt:tRNAs responsible for translating these proteins. Mitochondrial RNase P (mtRNase P) is a three-protein complex responsible for cleaving and processing the 5'-end of mt:tRNAs. Mutations in any of the three proteins can cause mitochondrial disease, as well as mutations in mitochondrial DNA. Great strides have been made in understanding the enzymology of mtRNase P; however, how the loss of each protein causes mitochondrial dysfunction and abnormal mt:tRNA processing in vivo has not been examined in detail. Here, we used Drosophila genetics to selectively remove each member of the complex in order to assess their specific contributions to mt:tRNA cleavage. Using this powerful model, we find differential effects on cleavage depending on which complex member is lost and which mt:tRNA is being processed. These data revealed in vivo subtleties of mtRNase P function that could improve understanding of human diseases.


Assuntos
Mitocôndrias/enzimologia , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/genética , Ribonuclease P/metabolismo , Alelos , Animais , Drosophila melanogaster/genética , Mitocôndrias/patologia , Mutação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo
5.
Elife ; 102021 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-34180399

RESUMO

Endonucleolytic removal of 5'-leader sequences from tRNA precursor transcripts (pre-tRNAs) by ribonuclease P (RNase P) is essential for protein synthesis. Beyond RNA-based RNase P enzymes, protein-only versions of the enzyme exert this function in various eukarya (there termed PRORPs) and in some bacteria (Aquifex aeolicus and close relatives); both enzyme types belong to distinct subgroups of the PIN domain metallonuclease superfamily. Homologs of Aquifex RNase P (HARPs) are also expressed in some other bacteria and many archaea, where they coexist with RNA-based RNase P and do not represent the main RNase P activity. Here, we solved the structure of the bacterial HARP from Halorhodospira halophila by cryo-electron microscopy, revealing a novel screw-like dodecameric assembly. Biochemical experiments demonstrate that oligomerization is required for RNase P activity of HARPs. We propose that the tRNA substrate binds to an extended spike-helix (SH) domain that protrudes from the screw-like assembly to position the 5'-end in close proximity to the active site of the neighboring dimer. The structure suggests that eukaryotic PRORPs and prokaryotic HARPs recognize the same structural elements of pre-tRNAs (tRNA elbow region and cleavage site). Our analysis thus delivers the structural and mechanistic basis for pre-tRNA processing by the prokaryotic HARP system.


Assuntos
Halorhodospira halophila/genética , Ribonuclease P/genética , Archaea/genética , Archaea/metabolismo , Bactérias/genética , Bactérias/metabolismo , Microscopia Crioeletrônica , Halorhodospira halophila/metabolismo , Ribonuclease P/metabolismo
6.
Antimicrob Agents Chemother ; 65(8): e0030021, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-33972249

RESUMO

RNase P is an essential enzyme responsible for tRNA 5'-end maturation. In most bacteria, the enzyme is a ribonucleoprotein consisting of a catalytic RNA subunit and a small protein cofactor termed RnpA. Several studies have reported small-molecule inhibitors directed against bacterial RNase P that were identified by high-throughput screenings. Using the bacterial RNase P enzymes from Thermotoga maritima, Bacillus subtilis, and Staphylococcus aureus as model systems, we found that such compounds, including RNPA2000 (and its derivatives), iriginol hexaacetate, and purpurin, induce the formation of insoluble aggregates of RnpA rather than acting as specific inhibitors. In the case of RNPA2000, aggregation was induced by Mg2+ ions. These findings were deduced from solubility analyses by microscopy and high-performance liquid chromatography (HPLC), RnpA-inhibitor co-pulldown experiments, detergent addition, and RnpA titrations in enzyme activity assays. Finally, we used a B. subtilis RNase P depletion strain, whose lethal phenotype could be rescued by a protein-only RNase P of plant origin, for inhibition zone analyses on agar plates. These cell-based experiments argued against RNase P-specific inhibition of bacterial growth by RNPA2000. We were also unable to confirm the previously reported nonspecific RNase activity of S. aureus RnpA itself. Our results indicate that high-throughput screenings searching for bacterial RNase P inhibitors are prone to the identification of "false positives" that are also termed pan-assay interference compounds (PAINS).


Assuntos
Ribonuclease P , Infecções Estafilocócicas , Bacillus subtilis/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , RNA Bacteriano , Ribonuclease P/metabolismo , Staphylococcus aureus/genética
7.
Nat Commun ; 12(1): 1007, 2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33579946

RESUMO

Plant viruses cause massive crop yield loss worldwide. Most plant viruses are RNA viruses, many of which contain a functional tRNA-like structure. RNase P has the enzymatic activity to catalyze the 5' maturation of precursor tRNAs. It is also able to cleave tRNA-like structures. However, RNase P enzymes only accumulate in the nucleus, mitochondria, and chloroplasts rather than cytosol where virus replication takes place. Here, we report a biotechnology strategy based on the re-localization of plant protein-only RNase P to the cytosol (CytoRP) to target plant viruses tRNA-like structures and thus hamper virus replication. We demonstrate the cytosol localization of protein-only RNase P in Arabidopsis protoplasts. In addition, we provide in vitro evidences for CytoRP to cleave turnip yellow mosaic virus and oilseed rape mosaic virus. However, we observe varied in vivo results. The possible reasons have been discussed. Overall, the results provided here show the potential of using CytoRP for combating some plant viral diseases.


Assuntos
Resistência à Doença/fisiologia , Ribonuclease P/genética , Ribonuclease P/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Vírus do Mosaico/genética , Vírus do Mosaico/metabolismo , Vírus de Plantas/genética , Protoplastos/metabolismo , Precursores de RNA/metabolismo , RNA de Transferência/genética , Ribonuclease P/química
8.
Mol Cell ; 81(4): 870-883.e10, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33453165

RESUMO

The series of RNA folding events that occur during transcription can critically influence cellular RNA function. Here, we present reconstructing RNA dynamics from data (R2D2), a method to uncover details of cotranscriptional RNA folding. We model the folding of the Escherichia coli signal recognition particle (SRP) RNA and show that it requires specific local structural fluctuations within a key hairpin to engender efficient cotranscriptional conformational rearrangement into the functional structure. All-atom molecular dynamics simulations suggest that this rearrangement proceeds through an internal toehold-mediated strand-displacement mechanism, which can be disrupted with a point mutation that limits local structural fluctuations and rescued with compensating mutations that restore these fluctuations. Moreover, a cotranscriptional folding intermediate could be cleaved in vitro by recombinant E. coli RNase P, suggesting potential cotranscriptional processing. These results from experiment-guided multi-scale modeling demonstrate that even an RNA with a simple functional structure can undergo complex folding and processing during synthesis.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/química , Dobramento de RNA , RNA Bacteriano/química , Ribonuclease P/química , Partícula de Reconhecimento de Sinal/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , RNA Bacteriano/metabolismo , Ribonuclease P/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo
9.
Nucleic Acids Res ; 49(3): 1784-1800, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33469651

RESUMO

We describe a synthetic riboswitch element that implements a regulatory principle which directly addresses an essential tRNA maturation step. Constructed using a rational in silico design approach, this riboswitch regulates RNase P-catalyzed tRNA 5'-processing by either sequestering or exposing the single-stranded 5'-leader region of the tRNA precursor in response to a ligand. A single base pair in the 5'-leader defines the regulatory potential of the riboswitch both in vitro and in vivo. Our data provide proof for prior postulates on the importance of the structure of the leader region for tRNA maturation. We demonstrate that computational predictions of ligand-dependent structural rearrangements can address individual maturation steps of stable non-coding RNAs, thus making them amenable as promising target for regulatory devices that can be used as functional building blocks in synthetic biology.


Assuntos
Processamento Pós-Transcricional do RNA , RNA de Transferência/metabolismo , Ribonuclease P/metabolismo , Riboswitch , Simulação por Computador , Escherichia coli/genética , Ligantes , RNA de Transferência/química , Ribonuclease P/química , Termodinâmica
10.
Plant Mol Biol ; 105(4-5): 463-482, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33474657

RESUMO

KEY MESSAGE: SCL3 inhibits transcriptional activity of IDD-DELLA complex by acting as a co-repressor and repression activity is enhanced in the presence of GAF1 in a TOPLESS-independent manner. GRAS [GIBBERELLIN-INSENSITIVE (GAI), REPRESSOR OF ga1-3 (RGA) and SCARECROW (SCR)] proteins are a family of plant-specific transcriptional regulators that play diverse roles in development and signaling. GRAS family DELLA proteins act as growth repressors by inhibiting gibberellin (GA) signaling in response to developmental and environmental cues. DELLAs also act as co-activators of transcription factor GAI-ASSOCIATED FACTOR1 (GAF1)/INDETERMINATE DOMAIN2 (IDD2), the GAF1-DELLA complex activating transcription of GAF1 target genes. GAF1 also interacts with TOPLESS (TPL), a transcriptional co-repressor, in the absence of DELLA, the GAF1-TPL complex repressing transcription of the target genes. SCARECROW-LIKE3 (SCL3), another member of the GRAS family, is thought to inhibit transcriptional activity of the IDD-DELLA complex through competitive interaction with IDD. Here, we also revealed that SCL3 inhibits transcriptional activation by the GAF1-DELLA complex via repression activity rather than via competitive inhibition of the GAF1-DELLA interaction. Moreover, the repression activity of SCL3 was enhanced by GAF1 in a TPL-independent manner. While the GRAS domain of DELLA has transcriptional activation activity, that of SCL3 has repression activity. SCL3 also inhibited transcriptional activity of GAF1-RGA fusion proteins. Results from the co-immunoprecipitation assays and the yeast three-hybrid assay suggested the possibility that SCL3 forms a ternary complex with GAF1 and DELLA. These findings provide important information on DELLA-regulated GA signaling and new insight into the transcriptional repression mechanism.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas Correpressoras/genética , Regulação da Expressão Gênica de Plantas , Giberelinas/metabolismo , Ribonuclease P/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Correpressoras/metabolismo , Immunoblotting , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonuclease P/metabolismo , Transdução de Sinais/genética , Técnicas do Sistema de Duplo-Híbrido
11.
Nat Biotechnol ; 39(3): 347-356, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33077962

RESUMO

RNA-protein interaction networks govern many biological processes but are difficult to examine comprehensively. We devised ribonucleoprotein networks analyzed by mutational profiling (RNP-MaP), a live-cell chemical probing strategy that maps cooperative interactions among multiple proteins bound to single RNA molecules at nucleotide resolution. RNP-MaP uses a hetero-bifunctional crosslinker to freeze interacting proteins in place on RNA and then maps multiple bound proteins on single RNA strands by read-through reverse transcription and DNA sequencing. RNP-MaP revealed that RNase P and RMRP, two sequence-divergent but structurally related non-coding RNAs, share RNP networks and that network hubs define functional sites in these RNAs. RNP-MaP also identified protein interaction networks conserved between mouse and human XIST long non-coding RNAs and defined protein communities whose binding sites colocalize and form networks in functional regions of XIST. RNP-MaP enables discovery and efficient validation of functional protein interaction networks on long RNAs in living cells.


Assuntos
Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Humanos , Mapas de Interação de Proteínas , RNA Longo não Codificante/metabolismo , Reprodutibilidade dos Testes , Ribonuclease P/metabolismo
12.
Methods Mol Biol ; 2167: 147-169, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32712919

RESUMO

Kink-turns are important RNA structural modules that facilitate long-range tertiary interactions and form binding sites for members of the L7Ae family of proteins. Present in a wide variety of functional RNAs, kink-turns play key organizational roles in many RNA-based cellular processes, including translation, modification, and tRNA biogenesis. It is important to determine the contribution of kink-turns to the overall architecture of resident RNAs, as these modules dictate ribonucleoprotein (RNP) assembly and function. This chapter describes a site-directed, hydroxyl radical-mediated footprinting strategy that utilizes L7Ae-tethered chemical nucleases to experimentally validate computationally identified kink-turns in any RNA and under a wide variety of conditions. The work plan described here uses the catalytic RNase P RNA as an example to provide a blueprint for using this footprinting method to map RNA-protein interactions in other RNP complexes.


Assuntos
Proteínas Arqueais/química , Pegada de DNA/métodos , Ácido Edético/análogos & derivados , Radical Hidroxila/química , Dobramento de RNA/genética , RNA/química , Ribonuclease P/metabolismo , Sítios de Ligação , Ácido Edético/química , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , Ligação Proteica , RNA Catalítico/genética , RNA Catalítico/metabolismo , Transcrição Reversa , Ribonuclease P/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Análise de Sequência de DNA
13.
J Am Chem Soc ; 142(44): 18735-18740, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33095984

RESUMO

Capturing the folding dynamics of large, functionally important RNAs has relied primarily on global measurements of structure or on per-nucleotide chemical probing. These approaches infer, but do not directly measure, through-space structural interactions. Here we introduce trimethyloxonium (TMO) as a chemical probe for RNA. TMO alkylates RNA at high levels in seconds, and thereby enables time-resolved, single-molecule, through-space probing of RNA folding using the RING-MaP correlated chemical probing framework. Time-resolved correlations in the RNase P RNA-a functional RNA with a complex structure stabilized by multiple noncanonical interactions-revealed that a long-range tertiary interaction guides native RNA folding for both secondary and tertiary structure. This unanticipated nonhierarchical folding mechanism was directly validated by examining the consequences of concise disruption of the through-space interaction. Single-molecule, time-resolved RNA structure probing with TMO is poised to reveal a wide range of dynamic RNA folding processes and principles of RNA folding.


Assuntos
Oniocompostos/química , RNA/química , Alquilação , Pareamento de Bases , Conformação de Ácido Nucleico , RNA/metabolismo , Dobramento de RNA , Ribonuclease P/metabolismo , Ésteres do Ácido Sulfúrico/química
14.
Aging (Albany NY) ; 12(16): 16155-16171, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32702667

RESUMO

BACKGROUND: Old age has been demonstrated to be a risk factor for GBM, but the underlying biological mechanism is still unclear. We designed this study intending to determine a mechanistic explanation for the link between age and pathogenesis in GBM. RESULTS: The expression of RPP30, an independent prognostic factor in GBM, was negatively correlated with age in both tumor and non-tumor brain samples. However, the post-transcriptional modifications carried out by RPP30 were different in primary GBM and non-tumor brain samples. RPP30 affected protein expression of cancer pathways by performing RNA modifications. Further, we found that RPP30 was related to drug metabolism pathways important in GBM. The decreased expression of RPP30 in older patients might be a pathogenic factor for GBM. CONCLUSION: This study revealed the role of RPP30 in gliomagenesis and provided the theoretical foundation for targeted therapy. METHODS: In total, 616 primary GBM samples and 41 non-tumor brain samples were enrolled in this study. Transcriptome data and clinical information were obtained from the CGGA, TCGA, and GSE53890 databases. Gene Set Variation Analysis and Gene Ontology analyses were the primary analytical methods used in this study. All statistical analyses were performed using R.


Assuntos
Autoantígenos/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/enzimologia , Glioblastoma/enzimologia , Ribonuclease P/metabolismo , Fatores Etários , Autoantígenos/genética , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular , Proliferação de Células , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Mapas de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Ribonuclease P/genética , Transdução de Sinais , Transcriptoma
15.
Nucleic Acids Res ; 48(21): 11815-11826, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-32719843

RESUMO

Pentatricopeptide repeat (PPR) motifs are α-helical structures known for their modular recognition of single-stranded RNA sequences with each motif in a tandem array binding to a single nucleotide. Protein-only RNase P 1 (PRORP1) in Arabidopsis thaliana is an endoribonuclease that uses its PPR domain to recognize precursor tRNAs (pre-tRNAs) as it catalyzes removal of the 5'-leader sequence from pre-tRNAs with its NYN metallonuclease domain. To gain insight into the mechanism by which PRORP1 recognizes tRNA, we determined a crystal structure of the PPR domain in complex with yeast tRNAPhe at 2.85 Å resolution. The PPR domain of PRORP1 bound to the structurally conserved elbow of tRNA and recognized conserved structural features of tRNAs using mechanisms that are different from the established single-stranded RNA recognition mode of PPR motifs. The PRORP1 PPR domain-tRNAPhe structure revealed a conformational change of the PPR domain upon tRNA binding and moreover demonstrated the need for pronounced overall flexibility in the PRORP1 enzyme conformation for substrate recognition and catalysis. The PRORP1 PPR motifs have evolved strategies for protein-tRNA interaction analogous to tRNA recognition by the RNA component of ribonucleoprotein RNase P and other catalytic RNAs, indicating convergence on a common solution for tRNA substrate recognition.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/genética , Precursores de RNA/química , Ribonuclease P/química , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Clonagem Molecular , Sequência Conservada , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonuclease P/genética , Ribonuclease P/metabolismo , Alinhamento de Sequência , Especificidade por Substrato
16.
Nat Commun ; 11(1): 2173, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358529

RESUMO

RNase P and MRP are highly conserved, multi-protein/RNA complexes with essential roles in processing ribosomal and tRNAs. Three proteins found in both complexes, Pop1, Pop6, and Pop7 are also telomerase-associated. Here, we determine how temperature sensitive POP1 and POP6 alleles affect yeast telomerase. At permissive temperatures, mutant Pop1/6 have little or no effect on cell growth, global protein levels, the abundance of Est1 and Est2 (telomerase proteins), and the processing of TLC1 (telomerase RNA). However, in pop mutants, TLC1 is more abundant, telomeres are short, and TLC1 accumulates in the cytoplasm. Although Est1/2 binding to TLC1 occurs at normal levels, Est1 (and hence Est3) binding is highly unstable. We propose that Pop-mediated stabilization of Est1 binding to TLC1 is a pre-requisite for formation and nuclear localization of the telomerase holoenzyme. Furthermore, Pop proteins affect TLC1 and the RNA subunits of RNase P/MRP in very different ways.


Assuntos
Ribonuclease P/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Telomerase/metabolismo , Telômero/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Metilação , Ligação Proteica , RNA/metabolismo , Processamento de Terminações 3' de RNA/genética , Ribonuclease P/genética , Ribonucleoproteínas/genética , Proteínas de Saccharomyces cerevisiae/genética , Telomerase/genética , Telômero/química
17.
BMC Bioinformatics ; 21(Suppl 2): 78, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32164523

RESUMO

BACKGROUND: Finding the tumor location in the prostate is an essential pathological step for prostate cancer diagnosis and treatment. The location of the tumor - the laterality - can be unilateral (the tumor is affecting one side of the prostate), or bilateral on both sides. Nevertheless, the tumor can be overestimated or underestimated by standard screening methods. In this work, a combination of efficient machine learning methods for feature selection and classification are proposed to analyze gene activity and select them as relevant biomarkers for different laterality samples. RESULTS: A data set that consists of 450 samples was used in this study. The samples were divided into three laterality classes (left, right, bilateral). The aim of this work is to understand the genomic activity in each class and find relevant genes as indicators for each class with nearly 99% accuracy. The system identified groups of differentially expressed genes (RTN1, HLA-DMB, MRI1) that are able to differentiate samples among the three classes. CONCLUSION: The proposed method was able to detect sets of genes that can identify different laterality classes. The resulting genes are found to be strongly correlated with disease progression. HLA-DMB and EIF4G2, which are detected in the set of genes can detect the left laterality, were reported earlier to be in the same pathway called Allograft rejection SuperPath.


Assuntos
Regulação Neoplásica da Expressão Gênica , Aprendizado de Máquina , Neoplasias da Próstata/patologia , Área Sob a Curva , Autoantígenos/genética , Autoantígenos/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Humanos , Imageamento por Ressonância Magnética , Masculino , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Próstata/diagnóstico por imagem , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/genética , Curva ROC , Ribonuclease P/genética , Ribonuclease P/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo
18.
Commun Biol ; 3(1): 147, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32221480

RESUMO

RNA modifications affect the stability and function of RNA species, regulating important downstream processes. Modification levels are often dynamic, varying between tissues and individuals, although it is not always clear what modulates this or what impact it has on biological systems. Here, we quantify variation in m1A/G RNA modification levels at functionally important positions in the human mitochondrial genome across 11,552 samples from 39 tissue/cell types and find that modification levels are associated with mitochondrial transcript processing. We identify links between mitochondrial RNA modification levels and genetic variants in the nuclear genome, including a missense mutation in LONP1, and find that genetic variants within MRPP3 and TRMT61B are associated with RNA modification levels across a large number of tissues. Genetic variants linked to RNA modification levels are associated with multiple disease/disease-related phenotypes, including blood pressure, breast cancer and psoriasis, suggesting a role for mitochondrial RNA modification in complex disease.


Assuntos
Adenosina/análogos & derivados , Núcleo Celular/genética , Guanina/análogos & derivados , Mitocôndrias/genética , Processamento Pós-Transcricional do RNA , RNA Mitocondrial/genética , Proteases Dependentes de ATP/genética , Proteases Dependentes de ATP/metabolismo , Adenosina/metabolismo , Núcleo Celular/metabolismo , Bases de Dados Genéticas , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Guanina/metabolismo , Humanos , Metilação , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação de Sentido Incorreto , Fenótipo , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , RNA Mitocondrial/metabolismo , RNA-Seq , Ribonuclease P/genética , Ribonuclease P/metabolismo , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismo
19.
Artigo em Inglês | MEDLINE | ID: mdl-32039645

RESUMO

The modular structure of bacterial ribonuclease P (RNase P) ribozymes, which recognize tertiary structures of precursor tRNAs (pre-tRNAs) to cleave their 5' leader sequence, can be dissected physically into the two structured domain RNAs (S-domain and C-domain). Separately prepared S-domain RNA and C-domain RNA assemble to form bimolecular forms of RNase P ribozymes. We analyzed the effects of polyethylene glycols (PEGs) on pre-tRNA cleavage catalyzed by bimolecular RNase P ribozymes to examine the effects of molecular crowding on the reaction. PEG molecular crowders significantly enhanced the activities of bimolecular RNase P ribozymes, some of which were hardly active without PEGs.


Assuntos
Bacillus subtilis/enzimologia , Escherichia coli/enzimologia , Polietilenoglicóis/metabolismo , Ribonuclease P/metabolismo , Biocatálise , Estrutura Molecular , Polietilenoglicóis/química , RNA Bacteriano/biossíntese , RNA Bacteriano/química , Ribonuclease P/química
20.
Nucleic Acids Res ; 48(5): 2564-2578, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31993626

RESUMO

Ribonuclease P (RNase P) is essential for the 5'-end maturation of tRNAs in all kingdoms of life. In Escherichia coli, temperature sensitive mutations in either its protein (rnpA49) and or RNA (rnpB709) subunits lead to inviability at nonpermissive temperatures. Using the rnpA49 temperature sensitive allele, which encodes a partially defective RNase P at the permissive temperature, we show here for the first time that the processing of RNase P-dependent polycistronic tRNA operons to release pre-tRNAs is the essential function of the enzyme, since the majority of 5'-immature tRNAs can be aminoacylated unless their 5'-extensions ≥8 nt. Surprisingly, the failure of 5'-end maturation elicits increased polyadenylation of some pre-tRNAs by poly(A) polymerase I (PAP I), which exacerbates inviability. The absence of PAP I led to improved aminoacylation of 5'-immature tRNAs. Our data suggest a more dynamic role for PAP I in maintaining functional tRNA levels in the cell.


Assuntos
Escherichia coli/enzimologia , Escherichia coli/genética , Óperon/genética , Precursores de RNA/biossíntese , Ribonuclease P/metabolismo , Aminoacilação , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Mutação/genética , Poli A/metabolismo , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
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