RESUMO
Effective clearance of an infection requires that the immune system rapidly detects and neutralizes invading parasites while strictly avoiding self-antigens that would result in autoimmunity. The cellular machinery and complex signaling pathways that coordinate an effective immune response have generally been considered properties of the eukaryotic immune system. However, a surprisingly sophisticated adaptive immune system that relies on small RNAs for sequence-specific targeting of foreign nucleic acids was recently discovered in bacteria and archaea. Molecular vaccination in prokaryotes is achieved by integrating short fragments of foreign nucleic acids into a repetitive locus in the host chromosome known as a CRISPR (clustered regularly interspaced short palindromic repeat). Here we review the mechanisms of CRISPR-mediated immunity and discuss the ecological and evolutionary implications of these adaptive defense systems.
Assuntos
Imunidade Adaptativa/genética , Archaea/imunologia , Bactérias/imunologia , Sequências Repetidas Invertidas/genética , RNA Arqueal/genética , RNA Bacteriano/genética , Transdução de Sinais/genética , Archaea/genética , Bactérias/genética , Sequências Repetidas Invertidas/imunologia , RNA Arqueal/imunologia , RNA Bacteriano/imunologia , Transdução de Sinais/imunologiaRESUMO
Post-transcriptional modifications have critical roles in tRNA stability and function1-4. In thermophiles, tRNAs are heavily modified to maintain their thermal stability under extreme growth temperatures5,6. Here we identified 2'-phosphouridine (Up) at position 47 of tRNAs from thermophilic archaea. Up47 confers thermal stability and nuclease resistance to tRNAs. Atomic structures of native archaeal tRNA showed a unique metastable core structure stabilized by Up47. The 2'-phosphate of Up47 protrudes from the tRNA core and prevents backbone rotation during thermal denaturation. In addition, we identified the arkI gene, which encodes an archaeal RNA kinase responsible for Up47 formation. Structural studies showed that ArkI has a non-canonical kinase motif surrounded by a positively charged patch for tRNA binding. A knockout strain of arkI grew slowly at high temperatures and exhibited a synthetic growth defect when a second tRNA-modifying enzyme was depleted. We also identified an archaeal homologue of KptA as an eraser that efficiently dephosphorylates Up47 in vitro and in vivo. Taken together, our findings show that Up47 is a reversible RNA modification mediated by ArkI and KptA that fine-tunes the structural rigidity of tRNAs under extreme environmental conditions.
Assuntos
Archaea , RNA de Transferência , Termotolerância , Archaea/genética , Ambientes Extremos , Fosforilação , Processamento Pós-Transcricional do RNA , RNA Arqueal/química , RNA Arqueal/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , UridinaRESUMO
Advances in genome-wide sequence technologies allow for detailed insights into the complexity of RNA landscapes of organisms from all three domains of life. Recent analyses of archaeal transcriptomes identified interaction and regulation networks of noncoding RNAs in this understudied domain. Here, we review current knowledge of small, noncoding RNAs with important functions for the archaeal lifestyle, which often requires adaptation to extreme environments. One focus is RNA metabolism at elevated temperatures in hyperthermophilic archaea, which reveals elevated amounts of RNA-guided RNA modification and virus defense strategies. Genome rearrangement events result in unique fragmentation patterns of noncoding RNA genes that require elaborate maturation pathways to yield functional transcripts. RNA-binding proteins, e.g., L7Ae and LSm, are important for many posttranscriptional control functions of RNA molecules in archaeal cells. We also discuss recent insights into the regulatory potential of their noncoding RNA partners.
Assuntos
Archaea/genética , Interação Gene-Ambiente , RNA Arqueal/genética , Pequeno RNA não Traduzido/genética , Archaea/metabolismo , Regulação da Expressão Gênica em Archaea , RNA Arqueal/metabolismo , Transdução de Sinais/genéticaRESUMO
N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA1-3. However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. Here we report ac4C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac4C at single-nucleotide resolution. In human and yeast mRNAs, ac4C sites are not detected but can be induced-at a conserved sequence motif-via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac4C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac4C and its potential thermoadaptive role. Our studies quantitatively define the ac4C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease4-6.
Assuntos
Acetilação , Citidina/análogos & derivados , Células Eucarióticas/metabolismo , Evolução Molecular , RNA/química , RNA/metabolismo , Archaea/química , Archaea/citologia , Archaea/genética , Archaea/crescimento & desenvolvimento , Sequência Conservada , Microscopia Crioeletrônica , Citidina/metabolismo , Células Eucarióticas/citologia , Células HeLa , Humanos , Modelos Moleculares , Acetiltransferases N-Terminal/metabolismo , RNA Arqueal/química , RNA Arqueal/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , TemperaturaRESUMO
Pseudouridine, one of the most abundant RNA modifications, is synthesized by stand-alone or RNA-guided pseudouridine synthases. Here, we comprehensively mapped pseudouridines in rRNAs, tRNAs and small RNAs in the archaeon Sulfolobus islandicus and identified Cbf5-associated H/ACA RNAs. Through genetic deletion and in vitro modification assays, we determined the responsible enzymes for these modifications. The pseudouridylation machinery in S. islandicus consists of the stand-alone enzymes aPus7 and aPus10, and six H/ACA RNA-guided enzymes that account for all identified pseudouridines. These H/ACA RNAs guide the modification of all eleven sites in rRNAs, two sites in tRNAs, and two sites in CRISPR RNAs. One H/ACA RNA shows exceptional versatility by targeting eight different sites. aPus7 and aPus10 are responsible for modifying positions 13, 54 and 55 in tRNAs. We identified four atypical H/ACA RNAs that lack the lower stem and the ACA motif and confirmed their function both in vivo and in vitro. Intriguingly, atypical H/ACA RNAs can be modified by Cbf5 in a guide-independent manner. Our data provide the first global view of pseudouridylation in archaea and reveal unexpected structures, substrates, and activities of archaeal H/ACA RNPs.
Assuntos
Pseudouridina , RNA Arqueal , RNA de Transferência , Sulfolobus , Pseudouridina/metabolismo , Sulfolobus/genética , Sulfolobus/metabolismo , RNA de Transferência/metabolismo , RNA de Transferência/genética , RNA Arqueal/genética , RNA Arqueal/metabolismo , RNA Arqueal/química , RNA Ribossômico/metabolismo , RNA Ribossômico/genética , Proteínas Arqueais/metabolismo , Proteínas Arqueais/genética , Processamento Pós-Transcricional do RNA , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismoRESUMO
Box C/D RNAs guide site-specific 2'-O-methylation of RNAs in archaea and eukaryotes. The defining feature of methylation guide RNAs is two sets of box C and D motifs that form kink-turn structures specifically recognized by L7Ae family proteins. Here, we engineered a new type of methylation guide that lacks C/D motifs and requires no L7Ae for assembly and function. We determined a crystal structure of a bipartite C/D-free guide RNA in complex with Nop5, fibrillarin and substrate in the active form at 2.2 Å resolution. The stems of new guide RNAs functionally replace C/D motifs in Nop5 binding, precisely placing the substrate for site-specific modification. We also found that the bipartite architecture and association of L7Ae with C/D motifs enhance modification when association of guide RNAs or substrates is weak. Our study provides insights into the variations, robustness and possible evolutionary path of methylation guide RNAs.
Assuntos
RNA Arqueal , RNA Guia de Cinetoplastídeos , RNA Arqueal/genética , RNA Guia de Cinetoplastídeos/genética , Metilação , Sequência de Bases , RNA/genética , RNA/metabolismo , RNA Nucleolar Pequeno/genética , Conformação de Ácido NucleicoRESUMO
The small CRISPR-derived RNAs of bacteria and archaea provide adaptive immunity by targeting the DNA of invading viruses and plasmids. Hale et al. (2009) now report on a new variant CRISPR/Cas complex in the archaeon Pyrococcus furiosus that uses guide RNAs to specifically target and cleave RNA not DNA.
Assuntos
Pyrococcus furiosus/genética , Pyrococcus furiosus/imunologia , Interferência de RNA , RNA Arqueal/imunologia , Pyrococcus furiosus/virologia , RNA Arqueal/genética , RNA Viral/imunologia , Pequeno RNA não TraduzidoRESUMO
Compelling evidence indicates that the CRISPR-Cas system protects prokaryotes from viruses and other potential genome invaders. This adaptive prokaryotic immune system arises from the clustered regularly interspaced short palindromic repeats (CRISPRs) found in prokaryotic genomes, which harbor short invader-derived sequences, and the CRISPR-associated (Cas) protein-coding genes. Here, we have identified a CRISPR-Cas effector complex that is comprised of small invader-targeting RNAs from the CRISPR loci (termed prokaryotic silencing (psi)RNAs) and the RAMP module (or Cmr) Cas proteins. The psiRNA-Cmr protein complexes cleave complementary target RNAs at a fixed distance from the 3' end of the integral psiRNAs. In Pyrococcus furiosus, psiRNAs occur in two size forms that share a common 5' sequence tag but have distinct 3' ends that direct cleavage of a given target RNA at two distinct sites. Our results indicate that prokaryotes possess a unique RNA silencing system that functions by homology-dependent cleavage of invader RNAs.
Assuntos
Proteínas Arqueais/imunologia , Pyrococcus furiosus/imunologia , Interferência de RNA , RNA Arqueal/imunologia , Proteínas Arqueais/metabolismo , Sequência de Bases , Pyrococcus furiosus/genética , Pyrococcus furiosus/metabolismo , Pyrococcus furiosus/virologia , RNA Arqueal/química , RNA Arqueal/genética , RNA Arqueal/metabolismo , RNA Viral/imunologia , Pequeno RNA não TraduzidoRESUMO
Posttranscriptional processes in Bacteria include the association of small regulatory RNAs (sRNA) with a target mRNA. The sRNA/mRNA annealing process is often mediated by an RNA chaperone called Hfq. The functional role of bacterial and eukaryotic Lsm proteins is partially understood, whereas knowledge about archaeal Lsm proteins is scarce. Here, we used the genetically tractable archaeal hyperthermophile Pyrococcus furiosus to identify the protein interaction partners of the archaeal Sm-like proteins (PfuSmAP1) using mass spectrometry and performed a transcriptome-wide binding site analysis of PfuSmAP1. Most of the protein interaction partners we found are part of the RNA homoeostasis network in Archaea including ribosomal proteins, the exosome, RNA-modifying enzymes, but also RNA polymerase subunits, and transcription factors. We show that PfuSmAP1 preferentially binds messenger RNAs and antisense RNAs recognizing a gapped poly(U) sequence with high affinity. Furthermore, we found that SmAP1 co-transcriptionally associates with target RNAs. Our study reveals that in contrast to bacterial Hfq, PfuSmAP1 does not affect the transcriptional activity or the pausing behaviour of archaeal RNA polymerases. We propose that PfuSmAP1 recruits antisense RNAs to target mRNAs and thereby executes its putative regulatory function on the posttranscriptional level.
Assuntos
Proteínas Arqueais , Pyrococcus furiosus , Pequeno RNA não Traduzido , Pyrococcus furiosus/genética , Pyrococcus furiosus/metabolismo , RNA Mensageiro/metabolismo , RNA Arqueal/genética , RNA Arqueal/química , RNA Arqueal/metabolismo , Sítios de Ligação , Bactérias/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Pequeno RNA não Traduzido/metabolismoRESUMO
The large ribosomal RNAs of eukaryotes frequently contain expansion sequences that add to the size of the rRNAs but do not affect their overall structural layout and are compatible with major ribosomal function as an mRNA translation machine. The expansion of prokaryotic ribosomal RNAs is much less explored. In order to obtain more insight into the structural variability of these conserved molecules, we herein report the results of a comprehensive search for the expansion sequences in prokaryotic 5S rRNAs. Overall, 89 expanded 5S rRNAs of 15 structural types were identified in 15 archaeal and 36 bacterial genomes. Expansion segments ranging in length from 13 to 109 residues were found to be distributed among 17 insertion sites. The strains harboring the expanded 5S rRNAs belong to the bacterial orders Clostridiales, Halanaerobiales, Thermoanaerobacterales, and Alteromonadales as well as the archael order Halobacterales When several copies of a 5S rRNA gene are present in a genome, the expanded versions may coexist with normal 5S rRNA genes. The insertion sequences are typically capable of forming extended helices, which do not seemingly interfere with folding of the conserved core. The expanded 5S rRNAs have largely been overlooked in 5S rRNA databases.
Assuntos
Genoma Arqueal , Genoma Bacteriano , RNA Arqueal/genética , RNA Bacteriano/genética , RNA Ribossômico 5S/genética , Alteromonadaceae/classificação , Alteromonadaceae/genética , Alteromonadaceae/metabolismo , Pareamento de Bases , Sequência de Bases , Clostridiales/classificação , Clostridiales/genética , Clostridiales/metabolismo , Firmicutes/classificação , Firmicutes/genética , Firmicutes/metabolismo , Halobacteriales/classificação , Halobacteriales/genética , Halobacteriales/metabolismo , Conformação de Ácido Nucleico , Filogenia , RNA Arqueal/química , RNA Arqueal/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Ribossômico 5S/química , RNA Ribossômico 5S/metabolismo , Thermoanaerobacterium/classificação , Thermoanaerobacterium/genética , Thermoanaerobacterium/metabolismoRESUMO
Bacteria and archaea rely on CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided adaptive immune systems for targeted elimination of foreign nucleic acids. These immune systems have been divided into three main types, and the first atomic-resolution structure of a type III RNA-guided immune complex provides new insights into the mechanisms of nucleic acid degradation. Here we compare the crystal structure of a type III complex to recently determined structures of DNA-targeting type I CRISPR complexes. Structural comparisons support previous assertions that type I and type III systems share a common ancestor and reveal how a conserved structural chassis is used to support RNA-, DNA-, or both RNA- and DNA-targeting mechanisms.
Assuntos
Archaea/virologia , Bactérias/virologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas Associadas a CRISPR/química , Modelos Moleculares , Filogenia , Ligação Proteica , Conformação Proteica , RNA Arqueal/química , RNA Arqueal/fisiologia , RNA Bacteriano/química , RNA Bacteriano/fisiologiaRESUMO
In prokaryotes, Clustered regularly interspaced short palindromic repeat (CRISPR)-derived RNAs (crRNAs), together with CRISPR-associated (Cas) proteins, capture and degrade invading genetic materials. In the type III-B CRISPR-Cas system, six Cas proteins (Cmr1-Cmr6) and a crRNA form an RNA silencing Cmr complex. Here we report the 2.1 Å crystal structure of the Cmr1-deficient, functional Cmr complex bound to single-stranded DNA, a substrate analog complementary to the crRNA guide. Cmr3 recognizes the crRNA 5' tag and defines the start position of the guide-target duplex, using its idiosyncratic loops. The ß-hairpins of three Cmr4 subunits intercalate within the duplex, causing nucleotide displacements with 6 nt intervals, and thus periodically placing the scissile bonds near the crucial aspartate of Cmr4. The structure reveals the mechanism for specifying the periodic target cleavage sites from the crRNA 5' tag and provides insights into the assembly of the type III interference machineries and the evolution of the Cmr and Cascade complexes.
Assuntos
Proteínas Arqueais/química , Proteínas Associadas a CRISPR/química , Interferência de RNA , RNA Arqueal/química , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Archaeoglobus fulgidus/genética , Archaeoglobus fulgidus/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Pyrococcus furiosus/genética , Pyrococcus furiosus/metabolismo , RNA Arqueal/genética , RNA Arqueal/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg2+-dependent cleavage of the 5' leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While â¼250-500 mM Mg2+ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg2+ requirement to â¼10-20 mM and improves the rate and fidelity of cleavage. To understand the Mg2+- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPRC domain and Cy5-RPRS domain), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg2+-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg2+ cooperativity. Our findings highlight how Mg2+ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.
Assuntos
Archaea/enzimologia , Magnésio/metabolismo , RNA Arqueal/genética , Ribonuclease P/genética , Conformação de Ácido Nucleico/efeitos dos fármacos , Pyrococcus furiosus/enzimologia , Pyrococcus furiosus/genética , Precursores de RNA/genética , RNA Arqueal/ultraestrutura , RNA Catalítico , Ribonuclease P/ultraestruturaRESUMO
Ribosomes are intricate molecular machines ensuring proper protein synthesis in every cell. Ribosome biogenesis is a complex process which has been intensively analyzed in bacteria and eukaryotes. In contrast, our understanding of the in vivo archaeal ribosome biogenesis pathway remains less characterized. Here, we have analyzed the in vivo role of the almost universally conserved ribosomal RNA dimethyltransferase KsgA/Dim1 homolog in archaea. Our study reveals that KsgA/Dim1-dependent 16S rRNA dimethylation is dispensable for the cellular growth of phylogenetically distant archaea. However, proteomics and functional analyses suggest that archaeal KsgA/Dim1 and its rRNA modification activity (i) influence the expression of a subset of proteins and (ii) contribute to archaeal cellular fitness and adaptation. In addition, our study reveals an unexpected KsgA/Dim1-dependent variability of rRNA modifications within the archaeal phylum. Combining structure-based functional studies across evolutionary divergent organisms, we provide evidence on how rRNA structure sequence variability (re-)shapes the KsgA/Dim1-dependent rRNA modification status. Finally, our results suggest an uncoupling between the KsgA/Dim1-dependent rRNA modification completion and its release from the nascent small ribosomal subunit. Collectively, our study provides additional understandings into principles of molecular functional adaptation, and further evolutionary and mechanistic insights into an almost universally conserved step of ribosome synthesis.
Assuntos
Archaea/enzimologia , Metiltransferases/metabolismo , RNA Arqueal/metabolismo , RNA Ribossômico/metabolismo , Archaea/genética , Movimento Celular , Crenarchaeota/enzimologia , Euryarchaeota/enzimologia , Haloferax volcanii/enzimologia , Metiltransferases/fisiologia , Biossíntese de Proteínas , RNA Arqueal/química , RNA Ribossômico/química , Subunidades Ribossômicas Menores de Arqueas/enzimologiaRESUMO
To improve and complete our knowledge of archaeal tRNA modification patterns, we have identified and compared the modification pattern (type and location) in tRNAs of three very different archaeal species, Methanococcus maripaludis (a mesophilic methanogen), Pyrococcus furiosus (a hyperthermophile thermococcale), and Sulfolobus acidocaldarius (an acidophilic thermophilic sulfolobale). Most abundant isoacceptor tRNAs (79 in total) for each of the 20 amino acids were isolated by two-dimensional gel electrophoresis followed by in-gel RNase digestions. The resulting oligonucleotide fragments were separated by nanoLC and their nucleotide content analyzed by mass spectrometry (MS/MS). Analysis of total modified nucleosides obtained from complete digestion of bulk tRNAs was also performed. Distinct base- and/or ribose-methylations, cytidine acetylations, and thiolated pyrimidines were identified, some at new positions in tRNAs. Novel, some tentatively identified, modifications were also found. The least diversified modification landscape is observed in the mesophilic Methanococcus maripaludis and the most complex one in Sulfolobus acidocaldarius Notable observations are the frequent occurrence of ac4C nucleotides in thermophilic archaeal tRNAs, the presence of m7G at positions 1 and 10 in Pyrococcus furiosus tRNAs, and the use of wyosine derivatives at position 37 of tRNAs, especially those decoding U1- and C1-starting codons. These results complete those already obtained by others with sets of archaeal tRNAs from Methanocaldococcus jannaschii and Haloferax volcanii.
Assuntos
Mathanococcus/genética , Nucleotídeos/química , Pyrococcus furiosus/genética , RNA de Transferência/química , RNA de Transferência/genética , Sulfolobus acidocaldarius/genética , Sequência de Bases , Conformação de Ácido Nucleico , RNA Arqueal/química , RNA Arqueal/genéticaRESUMO
Archaea and eukaryotes, in addition to protein-only enzymes, also possess ribonucleoproteins containing an H/ACA guide RNA plus four proteins that produce pseudouridine (Ψ). Although typical conditions for these RNA-guided reactions are known, certain variant conditions allow pseudouridylation. We used mutants of the two stem-loops of the Haloferax volcanii sR-h45 RNA that guides three pseudouridylations in 23S rRNA and their target RNAs to characterize modifications under various atypical conditions. The 5' stem-loop produces Ψ2605 and the 3' stem-loop produces Ψ1940 and Ψ1942. The latter two modifications require unpaired "UVUN" (V = A, C, or G) in the target and ACA box in the guide. Ψ1942 modification requires the presence of U1940 (or Ψ1940). Ψ1940 is not produced in the Ψ1942-containing substrate, suggesting a sequential modification of the two residues. The ACA box of a single stem-loop guide is not required when typically unpaired "UN" is up to 17 bases from its position in the guide, but is needed when the distance increases to 19 bases or the N is paired. However, ANA of the H box of the double stem-loop guide is needed even for the 5' typical pseudouridylation. The most 5' unpaired U in a string of U's is converted to Ψ, and in the absence of an unpaired U, a paired U can also be modified. Certain mutants of the Cbf5 protein affect pseudouridylation by the two stem-loops of sR-h45 differently. This study will help elucidate the conditions for production of nonconstitutive Ψ's, determine functions for orphan H/ACA RNAs and in target designing.
Assuntos
Pseudouridina/metabolismo , Processamento Pós-Transcricional do RNA , RNA Arqueal/genética , RNA Guia de Cinetoplastídeos/genética , Proteínas Arqueais/metabolismo , Haloferax volcanii/genética , Motivos de Nucleotídeos , Pseudouridina/química , RNA Arqueal/química , RNA Arqueal/metabolismo , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
General molecular principles of ribosome biogenesis have been well explored in bacteria and eukaryotes. Collectively, these studies have revealed important functional differences and few similarities between these processes. Phylogenetic studies suggest that the information processing machineries from archaea and eukaryotes are evolutionary more closely related than their bacterial counterparts. These observations raise the question of how ribosome synthesis in archaea may proceed in vivo. In this study, we describe a versatile plasmid-based cis-acting reporter system allowing to analyze in vivo the consequences of ribosomal RNA mutations in the model archaeon Haloferax volcanii. Applying this system, we provide evidence that the bulge-helix-bulge motif enclosed within the ribosomal RNA processing stems is required for the formation of archaeal-specific circular-pre-rRNA intermediates and mature rRNAs. In addition, we have collected evidences suggesting functional coordination of the early steps of ribosome synthesis in H. volcanii. Together our investigation describes a versatile platform allowing to generate and functionally analyze the fate of diverse rRNA variants, thereby paving the way to better understand the cis-acting molecular determinants necessary for archaeal ribosome synthesis, maturation, stability and function.
Assuntos
Redes Reguladoras de Genes/genética , Processamento Pós-Transcricional do RNA/genética , RNA Ribossômico/genética , Ribossomos/genética , Haloferax volcanii/genética , Mutação/genética , Filogenia , Precursores de RNA/genética , Estabilidade de RNA/genética , RNA Arqueal/genéticaRESUMO
tRNAs play a central role during the translation process and are heavily post-transcriptionally modified to ensure optimal and faithful mRNA decoding. These epitranscriptomics marks are added by largely conserved proteins and defects in the function of some of these enzymes are responsible for neurodevelopmental disorders and cancers. Here, we focus on the Trm11 enzyme, which forms N2-methylguanosine (m2G) at position 10 of several tRNAs in both archaea and eukaryotes. While eukaryotic Trm11 enzyme is only active as a complex with Trm112, an allosteric activator of methyltransferases modifying factors (RNAs and proteins) involved in mRNA translation, former studies have shown that some archaeal Trm11 proteins are active on their own. As these studies were performed on Trm11 enzymes originating from archaeal organisms lacking TRM112 gene, we have characterized Trm11 (AfTrm11) from the Archaeoglobus fulgidus archaeon, which genome encodes for a Trm112 protein (AfTrm112). We show that AfTrm11 interacts directly with AfTrm112 similarly to eukaryotic enzymes and that although AfTrm11 is active as a single protein, its enzymatic activity is strongly enhanced by AfTrm112. We finally describe the first crystal structures of the AfTrm11-Trm112 complex and of Trm11, alone or bound to the methyltransferase inhibitor sinefungin.
Assuntos
Proteínas Arqueais , Archaeoglobus fulgidus/enzimologia , RNA Arqueal/metabolismo , RNA de Transferência/metabolismo , tRNA Metiltransferases , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-Traducional , tRNA Metiltransferases/química , tRNA Metiltransferases/metabolismoRESUMO
A network of RNA helicases, endoribonucleases and exoribonucleases regulates the quantity and quality of cellular RNAs. To date, mechanistic studies focussed on bacterial and eukaryal systems due to the challenge of identifying the main drivers of RNA decay and processing in Archaea. Here, our data support that aRNase J, a 5'-3' exoribonuclease of the ß-CASP family conserved in Euryarchaeota, engages specifically with a Ski2-like helicase and the RNA exosome to potentially exert control over RNA surveillance, at the vicinity of the ribosome. Proteomic landscapes and direct protein-protein interaction analyses, strengthened by comprehensive phylogenomic studies demonstrated that aRNase J interplay with ASH-Ski2 and a cap exosome subunit. Finally, Thermococcus barophilus whole-cell extract fractionation experiments provide evidences that an aRNase J/ASH-Ski2 complex might exist in vivo and hint at an association of aRNase J with the ribosome that is emphasised in absence of ASH-Ski2. Whilst aRNase J homologues are found among bacteria, the RNA exosome and the Ski2-like RNA helicase have eukaryotic homologues, underlining the mosaic aspect of archaeal RNA machines. Altogether, these results suggest a fundamental role of ß-CASP RNase/helicase complex in archaeal RNA metabolism.
Assuntos
Euryarchaeota/enzimologia , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , RNA Helicases/metabolismo , Processamento Pós-Transcricional do RNA , RNA Arqueal/metabolismo , Mapeamento de Interação de Proteínas , Pyrococcus abyssi/enzimologia , Thermococcus/enzimologiaRESUMO
The exergonic reaction of FeS with H2S to form FeS2 (pyrite) and H2 was postulated to have operated as an early form of energy metabolism on primordial Earth. Since the Archean, sedimentary pyrite formation has played a major role in the global iron and sulfur cycles, with direct impact on the redox chemistry of the atmosphere. However, the mechanism of sedimentary pyrite formation is still being debated. We present microbial enrichment cultures which grew with FeS, H2S, and CO2 as their sole substrates to produce FeS2 and CH4 Cultures grew over periods of 3 to 8 mo to cell densities of up to 2 to 9 × 106 cells per mL-1 Transformation of FeS with H2S to FeS2 was followed by 57Fe Mössbauer spectroscopy and showed a clear biological temperature profile with maximum activity at 28 °C and decreasing activities toward 4 °C and 60 °C. CH4 was formed concomitantly with FeS2 and exhibited the same temperature dependence. Addition of either penicillin or 2-bromoethanesulfonate inhibited both FeS2 and CH4 production, indicating a coupling of overall pyrite formation to methanogenesis. This hypothesis was supported by a 16S rRNA gene-based phylogenetic analysis, which identified at least one archaeal and five bacterial species. The archaeon was closely related to the hydrogenotrophic methanogen Methanospirillum stamsii, while the bacteria were most closely related to sulfate-reducing Deltaproteobacteria, as well as uncultured Firmicutes and Actinobacteria. Our results show that pyrite formation can be mediated at ambient temperature through a microbially catalyzed redox process, which may serve as a model for a postulated primordial iron-sulfur world.