RESUMO
The RNase P family comprises structurally diverse endoribonucleases ranging from complex ribonucleoproteins to single polypeptides. We show that the organellar (AtPRORP1) and the two nuclear (AtPRORP2,3) single-polypeptide RNase P isoenzymes from Arabidopsis thaliana confer viability to Escherichia coli cells with a lethal knockdown of its endogenous RNA-based RNase P. RNA-Seq revealed that AtPRORP1, compared with bacterial RNase P or AtPRORP3, cleaves several precursor tRNAs (pre-tRNAs) aberrantly in E. coli. Aberrant cleavage by AtPRORP1 was mainly observed for pre-tRNAs that can form short acceptor-stem extensions involving G:C base pairs, including tRNAAsp(GUC), tRNASer(CGA) and tRNAHis. However, both AtPRORP1 and 3 were defective in processing of E. coli pre-tRNASec carrying an acceptor stem expanded by three G:C base pairs. Instead, pre-tRNASec was degraded, suggesting that tRNASec is dispensable for E. coli under laboratory conditions. AtPRORP1, 2 and 3 are also essentially unable to process the primary transcript of 4.5S RNA, a hairpin-like non-tRNA substrate processed by E. coli RNase P, indicating that PRORP enzymes have a narrower, more tRNA-centric substrate spectrum than bacterial RNA-based RNase P enzymes. The cells' viability also suggests that the essential function of the signal recognition particle can be maintained with a 5Î-extended 4.5S RNA.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Precursores de RNA/genética , Ribonuclease P/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Pareamento de Bases , Sequência de Bases , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Teste de Complementação Genética , Viabilidade Microbiana , Conformação de Ácido Nucleico , Precursores de RNA/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA de Transferência de Ácido Aspártico/genética , RNA de Transferência de Ácido Aspártico/metabolismo , RNA de Transferência de Histidina/genética , RNA de Transferência de Histidina/metabolismo , RNA de Transferência de Serina/genética , RNA de Transferência de Serina/metabolismo , Ribonuclease P/deficiência , Ribonuclease P/metabolismo , TransgenesRESUMO
RNase P is the enzyme that removes 5' extensions from tRNA precursors. With its diversity of enzyme forms-either protein- or RNA-based, ranging from single polypeptides to multi-subunit ribonucleoproteins-the RNase P enzyme family represents a unique model system to compare the evolution of enzymatic mechanisms. Here we present a comprehensive study of substrate recognition and cleavage-site selection by the nuclear single-subunit proteinaceous RNase P PRORP3 from Arabidopsis thaliana. Compared to bacterial RNase P, the best-characterized RNA-based enzyme form, PRORP3 requires a larger part of intact tRNA structure, but little to no determinants at the cleavage site or interactions with the 5' or 3' extensions of the tRNA. The cleavage site depends on the combined dimensions of acceptor stem and T domain, but also requires the leader to be single-stranded. Overall, the single-subunit PRORP appears mechanistically more similar to the complex nuclear ribonucleoprotein enzymes than to the simpler bacterial RNase P. Mechanistic similarity or dissimilarity among different forms of RNase P thus apparently do not necessarily reflect molecular composition or evolutionary relationship.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Precursores de RNA/química , RNA de Plantas/química , RNA de Transferência/química , Ribonuclease P/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Sequência de Bases , Clonagem Molecular , Sequência Conservada , Escherichia coli/genética , Escherichia coli/metabolismo , Evolução Molecular , Expressão Gênica , Isoenzimas/química , Isoenzimas/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Precursores de RNA/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA de Plantas/metabolismo , RNA de Transferência/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Ribonuclease P/genética , Especificidade por Substrato , Thermus thermophilus/enzimologia , Thermus thermophilus/genéticaRESUMO
MicroRNAs (miRNAs) represent a class of small non-coding RNAs that control gene expression by targeting mRNAs and triggering either repression of translation or RNA degradation. They have been shown to be involved in a variety of biological processes such as development, differentiation and cell cycle control, but little is known about their involvement in the response to irradiation. We showed here that in human umbilical vein endothelial cells (HUVEC) some miRNAs previously shown to have a crucial role in vascular biology are transiently modulated in response to a clinically relevant dose of ionizing radiation. In particular we identified an early transcriptional induction of several members of the microRNA cluster 17-92 and other microRNAs already known to be related to angiogenesis. At the same time we observed a peculiar behavior of the miR-221/222 cluster, suggesting an important role of these microRNAs in HUVEC homeostasis. We observed an increased efficiency in the formation of capillary-like structures in irradiated HUVEC. These results could lead to a new interpretation of the effect of ionizing radiation on endothelial cells and on the response of tumor endothelial bed cells to radiotherapy.