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
The regulation of mitochondrial RNA processing and its importance for ribosome biogenesis and energy metabolism are not clear. We generated conditional knockout mice of the endoribonuclease component of the RNase P complex, MRPP3, and report that it is essential for life and that heart and skeletal-muscle-specific knockout leads to severe cardiomyopathy, indicating that its activity is non-redundant. Transcriptome-wide parallel analyses of RNA ends (PARE) and RNA-seq enabled us to identify that in vivo 5' tRNA cleavage precedes 3' tRNA processing, and this is required for the correct biogenesis of the mitochondrial ribosomal subunits. We identify that mitoribosomal biogenesis proceeds co-transcriptionally because large mitoribosomal proteins can form a subcomplex on an unprocessed RNA containing the 16S rRNA. Taken together, our data show that RNA processing links transcription to translation via assembly of the mitoribosome.
Assuntos
Cardiomiopatias/genética , Ribossomos Mitocondriais/metabolismo , Biogênese de Organelas , Processamento Pós-Transcricional do RNA , Ribonuclease P/genética , Proteínas Ribossômicas/genética , Animais , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Fracionamento Celular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Músculo Esquelético , Miocárdio/metabolismo , Miocárdio/patologia , Biossíntese de Proteínas , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribonuclease P/deficiência , Proteínas Ribossômicas/metabolismo , Transcrição Gênica , TranscriptomaRESUMO
The universality of ribonuclease P (RNase P), the ribonucleoprotein essential for transfer RNA (tRNA) 5' maturation, is challenged in the archaeon Nanoarchaeum equitans. Neither extensive computational analysis of the genome nor biochemical tests in cell extracts revealed the existence of this enzyme. Here we show that the conserved placement of its tRNA gene promoters allows the synthesis of leaderless tRNAs, whose presence was verified by the observation of 5' triphosphorylated mature tRNA species. Initiation of tRNA gene transcription requires a purine, which coincides with the finding that tRNAs with a cytosine in position 1 display unusually extended 5' termini with an extra purine residue. These tRNAs were shown to be substrates for their cognate aminoacyl-tRNA synthetases. These findings demonstrate how nature can cope with the loss of the universal and supposedly ancient RNase P through genomic rearrangement at tRNA genes under the pressure of genome condensation.