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2.
Nucleic Acids Res ; 52(8): 4644-4658, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38375885

RESUMO

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/metabolismo
3.
Sci China Life Sci ; 67(4): 631-644, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38041781

RESUMO

Box C/D RNAs guide the site-specific formation of 2'-O-methylated nucleotides (Nm) of RNAs in eukaryotes and archaea. Although C/D RNAs have been profiled in several archaea, their targets have not been experimentally determined. Here, we mapped Nm in rRNAs, tRNAs, and abundant small RNAs (sRNAs) and profiled C/D RNAs in the crenarchaeon Sulfolobus islandicus. The targets of C/D RNAs were assigned by analysis of base-pairing interactions, in vitro modification assays, and gene deletion experiments, revealing a complicated landscape of C/D RNA-target interactions. C/D RNAs widely use dual antisense elements to target adjacent sites in rRNAs, enhancing modification at weakly bound sites. Two consecutive sites can be guided with the same antisense element upstream of box D or D', a phenomenon known as double-specificity that is exclusive to internal box D' in eukaryotic C/D RNAs. Several C/D RNAs guide modification at a single non-canonical site. This study reveals the global landscape of RNA-guided 2'-O-methylation in an archaeon and unexpected targeting rules employed by C/D RNA.


Assuntos
RNA Arqueal , RNA Guia de Sistemas CRISPR-Cas , Sequência de Bases , RNA Arqueal/genética , Conformação de Ácido Nucleico , RNA Ribossômico/genética
4.
Biol Chem ; 404(11-12): 1085-1100, 2023 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-37709673

RESUMO

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/metabolismo
5.
RNA ; 28(12): 1597-1605, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36127125

RESUMO

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 Nucleico
6.
Methods Mol Biol ; 2522: 243-254, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36125754

RESUMO

Posttranscriptional regulation actuated by small RNAs (sRNAs) plays essential roles in a wide variety of cellular processes, especially in stress responses and environmental signaling. Hundreds of sRNAs have recently been discovered in archaea using genome-wide approaches but the molecular mechanisms of only a few have been characterized experimentally. Here, we describe how to build sRNA sequencing libraries using size-selected total RNA in the model archaeon, Haloferax volcanii , to provide a tool to further characterize sRNAs in archaea.


Assuntos
Haloferax volcanii , Pequeno RNA não Traduzido , Biblioteca Gênica , Haloferax volcanii/genética , RNA Arqueal/genética , Pequeno RNA não Traduzido/genética , Análise de Sequência de RNA
7.
Nature ; 605(7909): 372-379, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35477761

RESUMO

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 , Uridina
8.
Nat Commun ; 13(1): 433, 2022 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-35064114

RESUMO

Replicative DNA polymerases cannot initiate DNA synthesis de novo and rely on dedicated RNA polymerases, primases, to generate a short primer. This primer is then extended by the DNA polymerase. In diverse archaeal species, the primase has long been known to have the ability to synthesize both RNA and DNA. However, the relevance of these dual nucleic acid synthetic modes for productive primer synthesis has remained enigmatic. In the current work, we reveal that the ability of primase to polymerize DNA serves dual roles in promoting the hand-off of the primer to the replicative DNA polymerase holoenzyme. First, it creates a 5'-RNA-DNA-3' hybrid primer which serves as an optimal substrate for elongation by the replicative DNA polymerase. Second, it promotes primer release by primase. Furthermore, modeling and experimental data indicate that primase incorporates a deoxyribonucleotide stochastically during elongation and that this switches the primase into a dedicated DNA synthetic mode polymerase.


Assuntos
DNA Primase/metabolismo , Primers do DNA/metabolismo , Replicação do DNA , DNA Arqueal/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , RNA Arqueal/biossíntese , Polarização de Fluorescência , Cinética , Modelos Biológicos , Nucleotídeos/metabolismo , Polimerização , Processos Estocásticos
10.
Nat Commun ; 12(1): 5281, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489402

RESUMO

The archaeal phylum Woesearchaeota, within the DPANN superphylum, includes phylogenetically diverse microorganisms that inhabit various environments. Their biology is poorly understood due to the lack of cultured isolates. Here, we analyze datasets of Woesearchaeota 16S rRNA gene sequences and metagenome-assembled genomes to infer global distribution patterns, ecological preferences and metabolic capabilities. Phylogenomic analyses indicate that the phylum can be classified into ten subgroups, termed A-J. While a symbiotic lifestyle is predicted for most, some members of subgroup J might be host-independent. The genomes of several Woesearchaeota, including subgroup J, encode putative [FeFe] hydrogenases (known to be important for fermentation in other organisms), suggesting that these archaea might be anaerobic fermentative heterotrophs.


Assuntos
Archaea/genética , Proteínas Arqueais/genética , Genoma Arqueal , Hidrogenase/genética , RNA Arqueal/genética , RNA Ribossômico 16S/genética , Sequência de Aminoácidos , Anaerobiose/genética , Archaea/classificação , Archaea/enzimologia , Proteínas Arqueais/metabolismo , Evolução Biológica , Fermentação , Processos Heterotróficos/genética , Hidrogenase/metabolismo , Metagenoma , Filogenia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
11.
Nucleic Acids Res ; 49(16): 9444-9458, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34387688

RESUMO

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 ∼250-500 mM Mg2+ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg2+ requirement to ∼10-20 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/ultraestrutura
12.
Science ; 372(6541)2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33926924

RESUMO

CRISPR-Cas systems provide RNA-guided adaptive immunity in prokaryotes. We report that the multisubunit CRISPR effector Cascade transcriptionally regulates a toxin-antitoxin RNA pair, CreTA. CreT (Cascade-repressed toxin) is a bacteriostatic RNA that sequesters the rare arginine tRNAUCU (transfer RNA with anticodon UCU). CreA is a CRISPR RNA-resembling antitoxin RNA, which requires Cas6 for maturation. The partial complementarity between CreA and the creT promoter directs Cascade to repress toxin transcription. Thus, CreA becomes antitoxic only in the presence of Cascade. In CreTA-deleted cells, cascade genes become susceptible to disruption by transposable elements. We uncover several CreTA analogs associated with diverse archaeal and bacterial CRISPR-cas loci. Thus, toxin-antitoxin RNA pairs can safeguard CRISPR immunity by making cells addicted to CRISPR-Cas, which highlights the multifunctionality of Cas proteins and the intricate mechanisms of CRISPR-Cas regulation.


Assuntos
Proteínas Associadas a CRISPR/fisiologia , Sistemas CRISPR-Cas/fisiologia , Haloarcula/fisiologia , RNA Arqueal/fisiologia , Sistemas Toxina-Antitoxina/fisiologia , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , Análise Mutacional de DNA , Regulação da Expressão Gênica em Archaea , Haloarcula/genética , Óperon , RNA de Transferência de Arginina/metabolismo , Sistemas Toxina-Antitoxina/genética
13.
RNA Biol ; 18(11): 1867-1881, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-33522404

RESUMO

While haloarchaea are highly resistant to oxidative stress, a comprehensive understanding of the processes regulating this remarkable response is lacking. Oxidative stress-responsive small non-coding RNAs (sRNAs) have been reported in the model archaeon, Haloferax volc anii, but targets and mechanisms have not been elucidated. Using a combination of high throughput and reverse molecular genetic approaches, we elucidated the functional role of the most up-regulated intergenic sRNA during oxidative stress in H. volcanii, named Small RNA in Haloferax Oxidative Stress (SHOxi). SHOxi was predicted to form a stable secondary structure with a conserved stem-loop region as the potential binding site for trans-targets. NAD-dependent malic enzyme mRNA, identified as a putative target of SHOxi, interacted directly with a putative 'seed' region within the predicted stem loop of SHOxi. Malic enzyme catalyzes the oxidative decarboxylation of malate into pyruvate using NAD+ as a cofactor. The destabilization of malic enzyme mRNA, and the decrease in the NAD+/NADH ratio, resulting from the direct RNA-RNA interaction between SHOxi and its trans-target was essential for the survival of H. volcanii to oxidative stress. These findings indicate that SHOxi likely regulates redox homoeostasis during oxidative stress by the post-transcriptional destabilization of malic enzyme mRNA. SHOxi-mediated regulation provides evidence that the fine-tuning of metabolic cofactors could be a core strategy to mitigate damage from oxidative stress and confer resistance. This study is the first to establish the regulatory effects of sRNAs on mRNAs during the oxidative stress response in Archaea.


Assuntos
Regulação da Expressão Gênica em Archaea , Regulação da Expressão Gênica , Haloferax volcanii/genética , RNA Antissenso/genética , RNA Arqueal/genética , RNA Mensageiro/genética , Pequeno RNA não Traduzido/genética , Homeostase , Oxirredução
14.
Nucleic Acids Res ; 49(3): 1662-1687, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33434266

RESUMO

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/enzimologia
15.
RNA Biol ; 18(10): 1382-1389, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33356826

RESUMO

Post-transcriptional RNA modifications play an important role in cellular metabolism with homoeostatic disturbances manifesting as a wide repertoire of phenotypes, reduced stress tolerance and translational perturbation, developmental defects, and diseases, such as type II diabetes, leukaemia, and carcinomas. Hence, there has been an intense effort to develop various methods for investigating RNA modifications and their roles in various organisms, including sequencing-based approaches and, more frequently, liquid chromatography-mass spectrometry (LC-MS)-based methods. Although LC-MS offers numerous advantages, such as being highly sensitive and quantitative over a broad detection range, some stationary phase chemistries struggle to resolve positional isomers. Furthermore, the demand for detailed analyses of complex biological samples often necessitates long separation times, hampering sample-to-sample turnover and making multisample analyses time consuming. To overcome this limitation, we have developed an ultra-performance LC-MS (UPLC-MS) method that uses an octadecyl carbon chain (C18)-bonded silica matrix for the efficient separation of 50 modified ribonucleosides, including positional isomers, in a single 9-min sample-to-sample run. To validate the performance and versatility of our method, we analysed tRNA modification patterns of representative microorganisms from each domain of life, namely Archaea (Methanosarcina acetivorans), Bacteria (Pseudomonas syringae), and Eukarya (Saccharomyces cerevisiae). Additionally, our method is flexible and readily applicable for detection and relative quantification using stable isotope labelling and targeted approaches like multiple reaction monitoring (MRM). In conclusion, this method represents a fast and robust tool for broad-range exploration and quantification of ribonucleosides, facilitating future homoeostasis studies of RNA modification in complex biological samples.


Assuntos
Methanosarcina/genética , Pseudomonas syringae/genética , RNA de Transferência/química , Ribonucleosídeos/análise , Saccharomyces cerevisiae/genética , Carbono/química , Cromatografia Líquida de Alta Pressão , Marcação por Isótopo , RNA Arqueal/genética , RNA Bacteriano/genética , RNA Fúngico/genética , Espectrometria de Massas em Tandem
16.
Sci China Life Sci ; 64(5): 678-696, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33140167

RESUMO

CRISPR-Cas systems provide the small RNA-based adaptive immunity to defend against invasive genetic elements in archaea and bacteria. Organisms of Sulfolobales, an order of thermophilic acidophiles belonging to the Crenarchaeotal Phylum, usually contain both type I and type III CRISPR-Cas systems. Two species, Saccharolobus solfataricus and Sulfolobus islandicus, have been important models for CRISPR study in archaea, and knowledge obtained from these studies has greatly expanded our understanding of molecular mechanisms of antiviral defense in all three steps: adaptation, expression and crRNA processing, and interference. Four subtypes of CRISPR-Cas systems are common in these organisms, including I-A, I-D, III-B, and III-D. These cas genes form functional modules, e.g., all genes required for adaptation and for interference in the I-A immune system are clustered together to form aCas and iCas modules. Genetic assays have been developed to study mechanisms of adaptation and interference by different CRISPR-Cas systems in these model archaea, and these methodologies are useful in demonstration of the protospacer-adjacent motif (PAM)-dependent DNA interference by I-A interference modules and multiple interference activities by III-B Cmr systems. Ribonucleoprotein effector complexes have been isolated for Sulfolobales III-B and III-D systems, and their biochemical characterization has greatly enriched the knowledge of molecular mechanisms of these novel antiviral immune responses.


Assuntos
Imunidade Adaptativa/genética , Sistemas CRISPR-Cas , Sulfolobales/genética , Sulfolobales/imunologia , DNA Arqueal/genética , Genes Arqueais , Modelos Biológicos , RNA Arqueal/genética , Transcrição Gênica
17.
RNA ; 27(2): 133-150, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33184227

RESUMO

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/metabolismo
18.
Biomolecules ; 10(12)2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33302546

RESUMO

Archaeal DNA polymerases from the B-family (polB) have found essential applications in biotechnology. In addition, some of their variants can accept a wide range of modified nucleotides or xenobiotic nucleotides, such as 1,5-anhydrohexitol nucleic acid (HNA), which has the unique ability to selectively cross-pair with DNA and RNA. This capacity is essential to allow the transmission of information between different chemistries of nucleic acid molecules. Variants of the archaeal polymerase from Thermococcus gorgonarius, TgoT, that can either generate HNA from DNA (TgoT_6G12) or DNA from HNA (TgoT_RT521) have been previously identified. To understand how DNA and HNA are recognized and selected by these two laboratory-evolved polymerases, we report six X-ray structures of these variants, as well as an in silico model of a ternary complex with HNA. Structural comparisons of the apo form of TgoT_6G12 together with its binary and ternary complexes with a DNA duplex highlight an ensemble of interactions and conformational changes required to promote DNA or HNA synthesis. MD simulations of the ternary complex suggest that the HNA-DNA hybrid duplex remains stable in the A-DNA helical form and help explain the presence of mutations in regions that would normally not be in contact with the DNA if it were not in the A-helical form. One complex with two incorporated HNA nucleotides is surprisingly found in a one nucleotide-backtracked form, which is new for a DNA polymerase. This information can be used for engineering a new generation of more efficient HNA polymerase variants.


Assuntos
Proteínas Arqueais/química , DNA Polimerase beta/química , DNA Arqueal/química , Hexosefosfatos/química , Nucleotídeos/química , RNA Arqueal/química , Thermococcus/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA Polimerase beta/genética , DNA Polimerase beta/metabolismo , DNA Arqueal/genética , DNA Arqueal/metabolismo , Evolução Molecular Direcionada/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Hexosefosfatos/metabolismo , Cinética , Simulação de Dinâmica Molecular , Mutação , Conformação de Ácido Nucleico , Nucleotídeos/genética , Nucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Engenharia de Proteínas/métodos , Domínios e Motivos de Interação entre Proteínas , RNA Arqueal/genética , RNA Arqueal/metabolismo , Especificidade por Substrato , Thermococcus/enzimologia
19.
BMC Genomics ; 21(1): 797, 2020 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-33198623

RESUMO

BACKGROUND: The archaeal exosome is an exoribonucleolytic multiprotein complex, which degrades single-stranded RNA in 3' to 5' direction phosphorolytically. In a reverse reaction, it can add A-rich tails to the 3'-end of RNA. The catalytic center of the exosome is in the aRrp41 subunit of its hexameric core. Its RNA-binding subunits aRrp4 and aDnaG confer poly(A) preference to the complex. The archaeal exosome was intensely characterized in vitro, but still little is known about its interaction with natural substrates in the cell, particularly because analysis of the transcriptome-wide interaction of an exoribonuclease with RNA is challenging. RESULTS: To determine binding sites of the exosome to RNA on a global scale, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) analysis with antibodies directed against aRrp4 and aRrp41 of the chrenarchaeon Sulfolobus solfataricus. A relatively high proportion (17-19%) of the obtained cDNA reads could not be mapped to the genome. Instead, they corresponded to adenine-rich RNA tails, which are post-transcriptionally synthesized by the exosome, and to circular RNAs (circRNAs). We identified novel circRNAs corresponding to 5' parts of two homologous, transposase-related mRNAs. To detect preferred substrates of the exosome, the iCLIP reads were compared to the transcript abundance using RNA-Seq data. Among the strongly enriched exosome substrates were RNAs antisense to tRNAs, overlapping 3'-UTRs and RNAs containing poly(A) stretches. The majority of the read counts and crosslink sites mapped in mRNAs. Furthermore, unexpected crosslink sites clustering at 5'-ends of RNAs was detected. CONCLUSIONS: In this study, RNA targets of an exoribonuclease were analyzed by iCLIP. The data documents the role of the archaeal exosome as an exoribonuclease and RNA-tailing enzyme interacting with all RNA classes, and underlines its role in mRNA turnover, which is important for adaptation of prokaryotic cells to changing environmental conditions. The clustering of crosslink sites near 5'-ends of genes suggests simultaneous binding of both RNA ends by the S. solfataricus exosome. This may serve to prevent translation of mRNAs dedicated to degradation in 3'-5' direction.


Assuntos
Proteínas Arqueais , Exossomos , Sulfolobus solfataricus , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Exossomos/genética , Exossomos/metabolismo , RNA/genética , Estabilidade de RNA , RNA Arqueal/genética , Sulfolobus solfataricus/genética , Sulfolobus solfataricus/metabolismo
20.
Nucleic Acids Res ; 48(19): 11068-11082, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33035335

RESUMO

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/metabolismo
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