RESUMEN
The endoribonuclease RNase P is responsible for tRNA 5' maturation in all domains of life. A unique feature of RNase P is the variety of enzyme architectures, ranging from dual- to multi-subunit ribonucleoprotein forms with catalytic RNA subunits to protein-only enzymes, the latter occurring as single- or multi-subunit forms or homo-oligomeric assemblies. The protein-only enzymes evolved twice: a eukaryal protein-only RNase P termed PRORP and a bacterial/archaeal variant termed homolog of Aquifex RNase P (HARP); the latter replaced the RNA-based enzyme in a small group of thermophilic bacteria but otherwise coexists with the ribonucleoprotein enzyme in a few other bacteria as well as in those archaea that also encode a HARP. Here we summarize the history of the discovery of protein-only RNase P enzymes and review the state of knowledge on structure and function of bacterial HARPs and eukaryal PRORPs, including human mitochondrial RNase P as a paradigm of multi-subunit PRORPs. We also describe the phylogenetic distribution and evolution of PRORPs, as well as possible reasons for the spread of PRORPs in the eukaryal tree and for the recruitment of two additional protein subunits to metazoan mitochondrial PRORP. We outline potential applications of PRORPs in plant biotechnology and address diseases associated with mutations in human mitochondrial RNase P genes. Finally, we consider possible causes underlying the displacement of the ancient RNA enzyme by a protein-only enzyme in a small group of bacteria.
Asunto(s)
Evolución Molecular , Ribonucleasa P , Animales , Humanos , Archaea/enzimología , Archaea/genética , Bacterias/enzimología , Bacterias/genética , Filogenia , Ribonucleasa P/química , Ribonucleasa P/clasificación , Ribonucleasa P/genética , Ribonucleasa P/metabolismo , ARN CatalíticoRESUMEN
BACKGROUND: N6-methyladenosine (m6A) is the most abundant mRNA modification, and controls mRNA stability. m6A distribution varies considerably between and within species. Yet, it is unclear to what extent this variability is driven by changes in genetic sequences ('cis') or cellular environments ('trans') and via which mechanisms. RESULTS: Here we dissect the determinants governing RNA methylation via interspecies and intraspecies hybrids in yeast and mammalian systems, coupled with massively parallel reporter assays and m6A-QTL reanalysis. We find that m6A evolution and variability is driven primarily in 'cis', via two mechanisms: (1) variations altering m6A consensus motifs, and (2) variation impacting mRNA secondary structure. We establish that mutations impacting RNA structure - even when distant from an m6A consensus motif - causally dictate methylation propensity. Finally, we demonstrate that allele-specific differences in m6A levels lead to allele-specific changes in gene expression. CONCLUSIONS: Our findings define the determinants governing m6A evolution and diversity and characterize the consequences thereof on gene expression regulation.
Asunto(s)
Adenina/análogos & derivados , Regulación de la Expresión Génica , ARN , Animales , ARN/genética , Metilación , ARN Mensajero/metabolismo , Mamíferos/genéticaRESUMEN
One mechanism of particular interest to regulate mRNA fate post-transcriptionally is mRNA modification. Especially the extent of m1A mRNA methylation is highly discussed due to methodological differences. However, one single m1A site in mitochondrial ND5 mRNA was unanimously reported by different groups. ND5 is a subunit of complex I of the respiratory chain. It is considered essential for the coupling of oxidation and proton transport. Here we demonstrate that this m1A site might be involved in the pathophysiology of Alzheimer's disease (AD). One of the pathological hallmarks of this neurodegenerative disease is mitochondrial dysfunction, mainly induced by Amyloid ß (Aß). Aß mainly disturbs functions of complex I and IV of the respiratory chain. However, the molecular mechanism of complex I dysfunction is still not fully understood. We found enhanced m1A methylation of ND5 mRNA in an AD cell model as well as in AD patients. Formation of this m1A methylation is catalyzed by increased TRMT10C protein levels, leading to translation repression of ND5. As a consequence, here demonstrated for the first time, TRMT10C induced m1A methylation of ND5 mRNA leads to mitochondrial dysfunction. Our findings suggest that this newly identified mechanism might be involved in Aß-induced mitochondrial dysfunction.
Asunto(s)
Adenosina , Enfermedad de Alzheimer , Péptidos beta-Amiloides , Complejo I de Transporte de Electrón , Mitocondrias , ARN Mensajero , Humanos , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/genética , ARN Mensajero/metabolismo , Adenosina/metabolismo , Mitocondrias/metabolismo , Metilación , Complejo I de Transporte de Electrón/metabolismo , Complejo I de Transporte de Electrón/genética , Péptidos beta-Amiloides/metabolismo , Masculino , Femenino , Anciano , Metiltransferasas/metabolismo , Metiltransferasas/genética , Anciano de 80 o más Años , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genéticaRESUMEN
RNase P is the endonuclease responsible for the 5' processing of precursor tRNAs (pre-tRNAs). Unlike the single-subunit protein-only RNase P (PRORP) found in plants or protists, human mitochondrial RNase P is a multi-enzyme assembly that in addition to the homologous PRORP subunit comprises a methyltransferase (TRMT10C) and a dehydrogenase (SDR5C1) subunit; these proteins, but not their enzymatic activities, are required for efficient pre-tRNA cleavage. Here we report a kinetic analysis of the cleavage reaction by human PRORP and its interplay with TRMT10C-SDR5C1 including 12 different mitochondrial pre-tRNAs. Surprisingly, we found that PRORP alone binds pre-tRNAs with nanomolar affinity and can even cleave some of them at reduced efficiency without the other subunits. Thus, the ancient binding mode, involving the tRNA elbow and PRORP's PPR domain, appears basically retained by human PRORP, and its metallonuclease domain is in principle correctly folded and functional. Our findings support a model according to which the main function of TRMT10C-SDR5C1 is to direct PRORP's nuclease domain to the cleavage site, thereby increasing the rate and accuracy of cleavage. This functional dependence of human PRORP on an extra tRNA-binding protein complex likely reflects an evolutionary adaptation to the erosion of canonical structural features in mitochondrial tRNAs.
Asunto(s)
ARN de Transferencia , Ribonucleasa P , Humanos , Ribonucleasa P/metabolismo , Cinética , ARN de Transferencia/metabolismo , Precursores del ARN/metabolismo , Endonucleasas/metabolismoRESUMEN
N6-methyladenosine (m6A), a widespread destabilizing mark on mRNA, is non-uniformly distributed across the transcriptome, yet the basis for its selective deposition is unknown. Here, we propose that m6A deposition is not selective. Instead, it is exclusion based: m6A consensus motifs are methylated by default, unless they are within a window of â¼100 nt from a splice junction. A simple model which we extensively validate, relying exclusively on presence of m6A motifs and exon-intron architecture, allows in silico recapitulation of experimentally measured m6A profiles. We provide evidence that exclusion from splice junctions is mediated by the exon junction complex (EJC), potentially via physical occlusion, and that previously observed associations between exon-intron architecture and mRNA decay are mechanistically mediated via m6A. Our findings establish a mechanism coupling nuclear mRNA splicing and packaging with the covalent installation of m6A, in turn controlling cytoplasmic decay.
Asunto(s)
Empalme del ARN , Transcriptoma , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estabilidad del ARN , Exones/genéticaRESUMEN
Human mitochondrial RNase P (mt-RNase P) is responsible for 5' end processing of mitochondrial precursor tRNAs, a vital step in mitochondrial RNA maturation, and is comprised of three protein subunits: TRMT10C, SDR5C1 (HSD10), and PRORP. Pathogenic variants in TRMT10C and SDR5C1 are associated with distinct recessive or x-linked infantile onset disorders, resulting from defects in mitochondrial RNA processing. We report four unrelated families with multisystem disease associated with bi-allelic variants in PRORP, the metallonuclease subunit of mt-RNase P. Affected individuals presented with variable phenotypes comprising sensorineural hearing loss, primary ovarian insufficiency, developmental delay, and brain white matter changes. Fibroblasts from affected individuals in two families demonstrated decreased steady state levels of PRORP, an accumulation of unprocessed mitochondrial transcripts, and decreased steady state levels of mitochondrial-encoded proteins, which were rescued by introduction of the wild-type PRORP cDNA. In mt-tRNA processing assays performed with recombinant mt-RNase P proteins, the disease-associated variants resulted in diminished mitochondrial tRNA processing. Identification of disease-causing variants in PRORP indicates that pathogenic variants in all three subunits of mt-RNase P can cause mitochondrial dysfunction, each with distinct pleiotropic clinical presentations.
Asunto(s)
Alelos , Pleiotropía Genética , Mitocondrias/enzimología , ARN Mitocondrial/genética , ARN de Transferencia/genética , Ribonucleasa P/genética , Adulto , Femenino , Humanos , Masculino , LinajeRESUMEN
The TRM10 family of methyltransferases is responsible for the N1-methylation of purines at position 9 of tRNAs in Archaea and Eukarya. The human genome encodes three TRM10-type enzymes, of which only the mitochondrial TRMT10C was previously characterized in detail, whereas the functional significance of the two presumably nuclear enzymes TRMT10A and TRMT10B remained unexplained. Here we show that TRMT10A is m1G9-specific and methylates a subset of nuclear-encoded tRNAs, whilst TRMT10B is the first m1A9-specific tRNA methyltransferase found in eukaryotes and is responsible for the modification of a single nuclear-encoded tRNA. Furthermore, we show that the lack of G9 methylation causes a decrease in the steady-state levels of the initiator tRNAiMet-CAT and an alteration in its further post-transcriptional modification. Our work finally clarifies the function of TRMT10A and TRMT10B in vivo and provides evidence that the loss of TRMT10A affects the pool of cytosolic tRNAs required for protein synthesis.
Asunto(s)
Metiltransferasas/metabolismo , ARNt Metiltransferasas/metabolismo , Secuencia de Bases , Línea Celular , Humanos , Metilación , Metiltransferasas/deficiencia , Biosíntesis de Proteínas , Purinas/metabolismo , ARN de Transferencia/metabolismoRESUMEN
Ribosome biogenesis requires numerous trans-acting factors, some of which are deeply conserved. In Bacteria, the endoribonuclease YbeY is believed to be involved in 16S rRNA 3'-end processing and its loss was associated with ribosomal abnormalities. In Eukarya, YBEY appears to generally localize to mitochondria (or chloroplasts). Here we show that the deletion of human YBEY results in a severe respiratory deficiency and morphologically abnormal mitochondria as an apparent consequence of impaired mitochondrial translation. Reduced stability of 12S rRNA and the deficiency of several proteins of the small ribosomal subunit in YBEY knockout cells pointed towards a defect in mitochondrial ribosome biogenesis. The specific interaction of mitoribosomal protein uS11m with YBEY suggests that the latter helps to properly incorporate uS11m into the nascent small subunit in its late assembly stage. This scenario shows similarities with final stages of cytosolic ribosome biogenesis, and may represent a late checkpoint before the mitoribosome engages in translation.
Asunto(s)
Ribosomas Mitocondriales/metabolismo , Ribonucleasas/metabolismo , Respiración de la Célula/genética , Escherichia coli/genética , Expresión Génica , Células HEK293 , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , ARN Ribosómico/metabolismo , Ribonucleasas/genética , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismoRESUMEN
N6-methyladenosine (m6A) is the most abundant modification on mRNA and is implicated in critical roles in development, physiology, and disease. A major limitation has been the inability to quantify m6A stoichiometry and the lack of antibody-independent methodologies for interrogating m6A. Here, we develop MAZTER-seq for systematic quantitative profiling of m6A at single-nucleotide resolution at 16%-25% of expressed sites, building on differential cleavage by an RNase. MAZTER-seq permits validation and de novo discovery of m6A sites, calibration of the performance of antibody-based approaches, and quantitative tracking of m6A dynamics in yeast gametogenesis and mammalian differentiation. We discover that m6A stoichiometry is "hard coded" in cis via a simple and predictable code, accounting for 33%-46% of the variability in methylation levels and allowing accurate prediction of m6A loss and acquisition events across evolution. MAZTER-seq allows quantitative investigation of m6A regulation in subcellular fractions, diverse cell types, and disease states.
Asunto(s)
Adenosina/análogos & derivados , ARN Mensajero/química , Análisis de Secuencia de ARN/métodos , Adenosina/análisis , Adenosina/inmunología , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Animales , Anticuerpos/inmunología , Cromatografía Líquida de Alta Presión , Cuerpos Embrioides/metabolismo , Células Madre Embrionarias , Endorribonucleasas/metabolismo , Humanos , Meiosis , Metilación , Ratones , Motivos de Nucleótidos , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Espectrometría de Masas en TándemRESUMEN
The genetic alphabet consists of the four letters: C, A, G, and T in DNA and C,A,G, and U in RNA. Triplets of these four letters jointly encode 20 different amino acids out of which proteins of all organisms are built. This system is universal and is found in all kingdoms of life. However, bases in DNA and RNA can be chemically modified. In DNA, around 10 different modifications are known, and those have been studied intensively over the past 20 years. Scientific studies on DNA modifications and proteins that recognize them gave rise to the large field of epigenetic and epigenomic research. The outcome of this intense research field is the discovery that development, ageing, and stem-cell dependent regeneration but also several diseases including cancer are largely controlled by the epigenetic state of cells. Consequently, this research has already led to the first FDA approved drugs that exploit the gained knowledge to combat disease. In recent years, the ~150 modifications found in RNA have come to the focus of intense research. Here we provide a perspective on necessary and expected developments in the fast expanding area of RNA modifications, termed epitranscriptomics.
Asunto(s)
ADN de Neoplasias , Epigénesis Genética , Epigenómica/normas , Perfilación de la Expresión Génica/normas , Regulación Neoplásica de la Expresión Génica , Neoplasias , ARN Neoplásico , Transcriptoma , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Europa (Continente) , Perfilación de la Expresión Génica/métodos , Humanos , Neoplasias/genética , Neoplasias/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismoRESUMEN
Modifications on mRNA offer the potential of regulating mRNA fate post-transcriptionally. Recent studies suggested the widespread presence of N1-methyladenosine (m1A), which disrupts Watson-Crick base pairing, at internal sites of mRNAs. These studies lacked the resolution of identifying individual modified bases, and did not identify specific sequence motifs undergoing the modification or an enzymatic machinery catalysing them, rendering it challenging to validate and functionally characterize putative sites. Here we develop an approach that allows the transcriptome-wide mapping of m1A at single-nucleotide resolution. Within the cytosol, m1A is present in a low number of mRNAs, typically at low stoichiometries, and almost invariably in tRNA T-loop-like structures, where it is introduced by the TRMT6/TRMT61A complex. We identify a single m1A site in the mitochondrial ND5 mRNA, catalysed by TRMT10C, with methylation levels that are highly tissue specific and tightly developmentally controlled. m1A leads to translational repression, probably through a mechanism involving ribosomal scanning or translation. Our findings suggest that m1A on mRNA, probably because of its disruptive impact on base pairing, leads to translational repression, and is generally avoided by cells, while revealing one case in mitochondria where tight spatiotemporal control over m1A levels was adopted as a potential means of post-transcriptional regulation.
Asunto(s)
Adenosina/análogos & derivados , Citosol/metabolismo , Mitocondrias/genética , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN/química , ARN/metabolismo , Adenosina/metabolismo , Emparejamiento Base , Complejo I de Transporte de Electrón/biosíntesis , Complejo I de Transporte de Electrón/genética , Regulación de la Expresión Génica , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Metilación , Metiltransferasas/metabolismo , Proteínas Mitocondriales/biosíntesis , Proteínas Mitocondriales/genética , Especificidad de Órganos , Biosíntesis de Proteínas , ARN/genética , ARN Mensajero/genética , ARN Mitocondrial , ARN de Transferencia/metabolismo , Transcriptoma , ARNt Metiltransferasas/genética , ARNt Metiltransferasas/metabolismoRESUMEN
RNase P is an essential tRNA-processing enzyme in all domains of life. We identified an unknown type of protein-only RNase P in the hyperthermophilic bacterium Aquifex aeolicus: Without an RNA subunit and the smallest of its kind, the 23-kDa polypeptide comprises a metallonuclease domain only. The protein has RNase P activity in vitro and rescued the growth of Escherichia coli and Saccharomyces cerevisiae strains with inactivations of their more complex and larger endogenous ribonucleoprotein RNase P. Homologs of Aquifex RNase P (HARP) were identified in many Archaea and some Bacteria, of which all Archaea and most Bacteria also encode an RNA-based RNase P; activity of both RNase P forms from the same bacterium or archaeon could be verified in two selected cases. Bioinformatic analyses suggest that A. aeolicus and related Aquificaceae likely acquired HARP by horizontal gene transfer from an archaeon.
Asunto(s)
Archaea/enzimología , Bacterias/enzimología , Ribonucleasa P/metabolismo , Archaea/genética , Bacterias/genética , Transferencia de Gen Horizontal , Filogenia , Ribonucleasa P/genética , Ribonucleasa P/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Precursores del ARN/genética , Ribonucleasa P/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Emparejamiento Base , Secuencia de Bases , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Prueba de Complementación Genética , Viabilidad Microbiana , Conformación de Ácido Nucleico , Precursores del ARN/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN de Transferencia de Aspártico/genética , ARN de Transferencia de Aspártico/metabolismo , ARN de Transferencia de Histidina/genética , ARN de Transferencia de Histidina/metabolismo , ARN de Transferencia de Serina/genética , ARN de Transferencia de Serina/metabolismo , Ribonucleasa P/deficiencia , Ribonucleasa P/metabolismo , TransgenesRESUMEN
Ribonuclease P (RNase P) is the enzyme that endonucleolytically removes 5'-precursor sequences from tRNA transcripts in all domains of life. RNase P activities are either ribonucleoprotein (RNP) or protein-only RNase P (PRORP) enzymes, raising the question about the mechanistic strategies utilized by these architecturally different enzyme classes to catalyze the same type of reaction. Here, we analyzed the kinetics and cleavage-site selection by PRORP3 from Arabidopsis thaliana (AtPRORP3) using precursor tRNAs (pre-tRNAs) with individual modifications at the canonical cleavage site, with either Rp- or Sp-phosphorothioate, or 2'-deoxy, 2'-fluoro, 2'-amino, or 2'-O-methyl substitutions. We observed a small but robust rescue effect of Sp-phosphorothioate-modified pre-tRNA in the presence of thiophilic Cd2+ ions, consistent with metal-ion coordination to the (pro-)Sp-oxygen during catalysis. Sp-phosphorothioate, 2'-deoxy, 2'-amino, and 2'-O-methyl modification redirected the cleavage mainly to the next unmodified phosphodiester in the 5'-direction. Our findings are in line with the 2'-OH substituent at nucleotide -1 being involved in an H-bonding acceptor function. In contrast to bacterial RNase P, AtPRORP3 was found to be able to utilize the canonical and upstream cleavage site with similar efficiency (corresponding to reduced cleavage fidelity), and the two cleavage pathways appear less interdependent than in the bacterial RNA-based system.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Precursores del ARN/metabolismo , ARN de Transferencia/metabolismo , Ribonucleasa P/metabolismo , Catálisis , Cinética , Especificidad por SustratoRESUMEN
The removal of transcriptional 5' and 3' extensions is an essential step in tRNA biogenesis. In some bacteria, tRNA 5'- and 3'-end maturation require no further steps, because all their genes encode the full tRNA sequence. Often however, the ends are incomplete, and additional maturation, repair or editing steps are needed. In all Eukarya, but also many Archaea and Bacteria, e.g., the universal 3'-terminal CCA is not encoded and has to be added by the CCA-adding enzyme. Apart from such widespread "repair/maturation" processes, tRNA genes in some cases apparently cannot give rise to intact, functional tRNA molecules without further, more specific end repair or editing. Interestingly, the responsible enzymes as far as identified appear to be polymerases usually involved in regular tRNA repair after damage. Alternatively, enzymes are recruited from other non-tRNA pathways; e.g., in animal mitochondria, poly(A) polymerase plays a crucial role in the 3'-end repair/editing of tRNAs. While these repair/editing pathways apparently allowed peculiar tRNA-gene overlaps or mismatching mutations in the acceptor stem to become genetically fixed in some present-day organisms, they may have also driven some global changes in tRNA maturation on a greater evolutionary scale.
Asunto(s)
Archaea/genética , Bacterias/genética , ARN de Transferencia/metabolismo , Animales , Archaea/metabolismo , Bacterias/metabolismo , Evolución Molecular , Edición de ARN , Procesamiento Postranscripcional del ARN , ARN de Archaea/genética , ARN de Archaea/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN de Transferencia/química , ARN de Transferencia/genéticaRESUMEN
We report a Caucasian boy with intractable epilepsy and global developmental delay. Whole-exome sequencing identified the likely genetic etiology as a novel p.K212E mutation in the X-linked gene HSD17B10 for mitochondrial short-chain dehydrogenase/reductase SDR5C1. Mutations in HSD17B10 cause the HSD10 disease, traditionally classified as a metabolic disorder due to the role of SDR5C1 in fatty and amino acid metabolism. However, SDR5C1 is also an essential subunit of human mitochondrial RNase P, the enzyme responsible for 5'-processing and methylation of purine-9 of mitochondrial tRNAs. Here we show that the p.K212E mutation impairs the SDR5C1-dependent mitochondrial RNase P activities, and suggest that the pathogenicity of p.K212E is due to a general mitochondrial dysfunction caused by reduction in SDR5C1-dependent maturation of mitochondrial tRNAs.
Asunto(s)
3-Hidroxiacil-CoA Deshidrogenasas/genética , Discapacidades del Desarrollo/genética , Epilepsia Refractaria/genética , Mutación , Ribonucleasa P/metabolismo , Análisis de Secuencia de ADN/métodos , Niño , Exoma , Genes Ligados a X , Humanos , Masculino , Mitocondrias/genética , Mitocondrias/metabolismo , ARN de Transferencia/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/enzimología , Precursores del ARN/química , ARN de Planta/química , ARN de Transferencia/química , Ribonucleasa P/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Bacillus subtilis/enzimología , Bacillus subtilis/genética , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , Escherichia coli/genética , Escherichia coli/metabolismo , Evolución Molecular , Expresión Génica , Isoenzimas/química , Isoenzimas/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Precursores del ARN/metabolismo , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN de Planta/metabolismo , ARN de Transferencia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Ribonucleasa P/genética , Especificidad por Sustrato , Thermus thermophilus/enzimología , Thermus thermophilus/genéticaRESUMEN
RNase P is the endonuclease that removes 5' leader sequences from tRNA precursors. In Eukarya, separate RNase P activities exist in the nucleus and mitochondria/plastids. Although all RNase P enzymes catalyze the same reaction, the different architectures found in Eukarya range from ribonucleoprotein (RNP) enzymes with a catalytic RNA and up to 10 protein subunits to single-subunit protein-only RNase P (PRORP) enzymes. Here, analysis of the phylogenetic distribution of RNP and PRORP enzymes in Eukarya revealed 1) a wealth of novel P RNAs in previously unexplored phylogenetic branches and 2) that PRORP enzymes are more widespread than previously appreciated, found in four of the five eukaryal supergroups, in the nuclei and/or organelles. Intriguingly, the occurrence of RNP RNase P and PRORP seems mutually exclusive in genetic compartments of modern Eukarya. Our comparative analysis provides a global picture of the evolution and diversification of RNase P throughout Eukarya.