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1.
Planta ; 254(6): 116, 2021 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-34750674

RESUMO

MAIN CONCLUSION: The novel structural variations were identified in cotton chloroplast tRNAs and gene loss events were more obvious than duplications in chloroplast tRNAs. Transfer RNAs (tRNA) have long been believed an evolutionary-conserved molecular family, which play the key roles in the process of protein biosynthesis in plant life activities. In this study, we detected the evolutionary characteristics and phylogeny of chloroplast tRNAs in cotton plants, an economic and fibered important taxon in the world. We firstly annotated the chloroplast tRNAs of 27 Gossypium species to analyze their genetic composition, structural characteristics and evolution. Compared with the traditional view of evolutionary conservation of tRNA, some novel tRNA structural variations were identified in cotton plants. I.g., tRNAVal-UAC and tRNAIle-GAU only contained one intron in the anti-condon loop region of tRNA secondary structure, respectively. In the variable region, some tRNAs contained a circle structure with a few nucleotides. Interestingly, the calculation result of free energy indicated that the variation of novel tRNAs contributed to the stability of tRNA structure. Phylogenetic analysis suggested that chloroplast tRNAs have evolved from multiple common ancestors, and the tRNAMet seemed to be an ancestral tRNA, which can be duplicated and diversified to produce other tRNAs. The chloroplast tRNAs contained a group I intron in cotton plants, and the evolutionary analysis of introns indicated that group I intron of chloroplast tRNA originated from cyanobacteria. Analysis of gene duplication and loss events showed that gene loss events were more obvious than duplications in Gossypium chloroplast tRNAs. Additionally, we found that the rate of transition was higher than the ones of transversion in cotton chloroplast tRNAs. This study provided new insights into the structural characteristics and evolution of chloroplast tRNAs in cotton plants.


Assuntos
Evolução Molecular , Gossypium , Cloroplastos/genética , Gossypium/genética , Filogenia , RNA de Transferência/genética
2.
BMC Genomics ; 22(1): 716, 2021 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-34600469

RESUMO

BACKGROUND: Stomatopod crustaceans are aggressive marine predators featuring complex compound eyes and powerful raptorial appendages used for "smashing" or "spearing" prey and/or competitors. Among them, parasquilloids (superfamily Parasquilloidea) possess eyes with 2-3 midband rows of hexagonal ommatidia and spearing appendages. Here, we assembled and analyzed the complete mitochondrial genome of the parasquilloid Faughnia haani and explored family- and superfamily-level phylogenetic relationships within the Stomatopoda based on mitochondrial protein coding genes (PCGs). RESULTS: The mitochondrial genome of F. haani is 16,089 bp in length and encodes 13 protein coding genes (PCGs), 22 transfer RNA genes, 2 ribosomal RNA genes, and a control region that is relatively well organized, containing 2 GA-blocks, 4 poly-T stretches, various [TA(A)]n-blocks, and 2 hairpin structures. This organized control region is likely a synapomorphic characteristic in the Stomatopoda. Comparison of the control region among superfamilies shows that parasquilloid species are more similar to gonodactyloids than to squilloids and lysiosquilloids given the presence of various  poly-T stretches between the hairpin structures and [TA(A)]n-blocks. Synteny is identical to that reported for other stomatopods and corresponds to the Pancrustacea ground pattern. A maximum-likelihood phylogenetic tree based on PCGs revealed that Parasquilloidea is sister to Lysiosquilloidea and Gonodactyloidea and not to Squilloidea, contradicting previous phylogenetic studies. CONCLUSIONS: The novel phylogenetic position of Parasquilloidea revealed by our study indicates that 'spearing' raptorial appendages are plesiomorphic and that the 'smashing' type is either derived (as reported in previous studies) or apomorphic. Our results raise the possibility that the spearing raptorial claw may have independently evolved twice. The superfamily Parasquilloidea exhibits a closer relationship with other stomatopod superfamilies with a different raptorial claw type and with dissimilar numbers of midband rows of hexagonal ommatidia. Additional studies focusing on the assembly of mitochondrial genomes from species belonging to different genera, families, and superfamilies within the order Stomatopoda are warranted to reach a robust conclusion regarding the evolutionary history of this iconic clade based on mitochondrial PCGs.


Assuntos
Genoma Mitocondrial , Animais , Evolução Biológica , Crustáceos , Humanos , Filogenia , RNA de Transferência/genética
3.
BMC Genomics ; 22(1): 750, 2021 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-34663228

RESUMO

BACKGROUND: Chloroplast transfer RNAs (tRNAs) can participate in various vital processes. Gymnosperms have important ecological and economic value, and they are the dominant species in forest ecosystems in the Northern Hemisphere. However, the evolution and structural changes in chloroplast tRNAs in gymnosperms remain largely unclear. RESULTS: In this study, we determined the nucleotide evolution, phylogenetic relationships, and structural variations in 1779 chloroplast tRNAs in gymnosperms. The numbers and types of tRNA genes present in the chloroplast genomes of different gymnosperms did not differ greatly, where the average number of tRNAs was 33 and the frequencies of occurrence for various types of tRNAs were generally consistent. Nearly half of the anticodons were absent. Molecular sequence variation analysis identified the conserved secondary structures of tRNAs. About a quarter of the tRNA genes were found to contain precoded 3' CCA tails. A few tRNAs have undergone novel structural changes that are closely related to their minimum free energy, and these structural changes affect the stability of the tRNAs. Phylogenetic analysis showed that tRNAs have evolved from multiple common ancestors. The transition rate was higher than the transversion rate in gymnosperm chloroplast tRNAs. More loss events than duplication events have occurred in gymnosperm chloroplast tRNAs during their evolutionary process. CONCLUSIONS: These findings provide novel insights into the molecular evolution and biological characteristics of chloroplast tRNAs in gymnosperms.


Assuntos
Cycadopsida , Ecossistema , Cloroplastos/genética , Cycadopsida/genética , Filogenia , RNA de Transferência/genética
4.
BMC Genomics ; 22(1): 755, 2021 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-34674653

RESUMO

BACKGROUND: Mitochondrial genomes (mitogenomes) have greatly improved our understanding of the backbone phylogeny of Lepidoptera, but few studies on comparative mitogenomics below the family level have been conducted. Here, we generated 13 mitogenomes of eight tortricid species, reannotated 27 previously reported mitogenomes, and systematically performed a comparative analysis of nucleotide composition, gene variation and phylogenetic performance. RESULTS: The lengths of completely sequenced mitogenomes ranged from 15,440 bp to 15,778 bp, and the gene content and organization were conserved in Tortricidae and typical for Lepidoptera. Analyses of AT-skew and GC-skew, the effective number of codons and the codon bias index all show a base bias in Tortricidae, with little heterogeneity among the major tortricid groups. Variations in the divergence rates among 13 protein-coding genes of the same tortricid subgroup and of the same PCG among tortricid subgroups were detected. The secondary structures of 22 transfer RNA genes and two ribosomal RNA genes were predicted and comparatively illustrated, showing evolutionary heterogeneity among different RNAs or different regions of the same RNA. The phylogenetic uncertainty of Enarmoniini in Tortricidae was confirmed. The synonymy of Bactrini and Olethreutini was confirmed for the first time, with the representative Bactrini consistently nesting in the Olethreutini clade. Nad6 exhibits the highest phylogenetic informativeness from the root to the tip of the resulting tree, and the combination of the third coding positions of 13 protein-coding genes shows extremely high phylogenetic informativeness. CONCLUSIONS: This study presents 13 mitogenomes of eight tortricid species and represents the first detailed comparative mitogenomics study of Tortricidae. The results further our understanding of the evolutionary architectures of tortricid mitogenomes and provide a basis for future studies of population genetics and phylogenetic investigations in this group.


Assuntos
Genoma Mitocondrial , Mariposas , Animais , Mariposas/genética , Nucleotídeos/genética , Filogenia , RNA Ribossômico/genética , RNA de Transferência/genética
5.
Int J Mol Sci ; 22(19)2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34638533

RESUMO

The widespread implementation of mass sequencing has revealed a diverse landscape of small RNAs derived from larger precursors. Whilst many of these are likely to be byproducts of degradation, there are nevertheless metabolically stable fragments derived from tRNAs, rRNAs, snoRNAs, and other non-coding RNA, with a number of examples of the production of such fragments being conserved across species. Coupled with specific interactions to RNA-binding proteins and a growing number of experimentally reported examples suggesting function, a case is emerging whereby the biological significance of small non-coding RNAs extends far beyond miRNAs and piRNAs. Related to this, a similarly complex picture is emerging of non-canonical roles for the non-coding precursors, such as for snoRNAs that are also implicated in such areas as the silencing of gene expression and the regulation of alternative splicing. This is in addition to a body of literature describing snoRNAs as an additional source of miRNA-like regulators. This review seeks to highlight emerging roles for such non-coding RNA, focusing specifically on "new" roles for snoRNAs and the small fragments derived from them.


Assuntos
Processamento Alternativo/genética , Regulação da Expressão Gênica/genética , Processamento Pós-Transcricional do RNA/genética , RNA Nucleolar Pequeno/genética , Humanos , MicroRNAs/genética , Neoplasias/genética , RNA Nucleolar Pequeno/metabolismo , RNA de Transferência/genética , Proteínas de Ligação a RNA/metabolismo
6.
Int J Clin Oncol ; 26(12): 2229-2236, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34689291

RESUMO

OBJECTIVES: This study aimed to investigate the role of the tRNA aspartic acid methyltransferase 1 (TRDMT1) protein in the development and progression of gastric cancer (GC). METHODS: The 90 GC tissues and 35 paracancerous tissues (gastric mucosa) were collected from patients (31 males and 59 females; average age 66), who were pathologically diagnosed as GC. The expression of TRDMT1 in three GC cell lines (MKN28, BGC823, and MGC803) and tissues from GC patients were detected by western blotting and immunological staining, respectively. The relationship between TRDMT1 expression and clinicopathological parameters in GC patients was explored. TRDMT1 was knocked down by RNAi lentivirus in GC cells. GC cell migration and invasion were analyzed using scratch and transwell assays. RESULTS: TRDMT1 expression in the GC cell lines was higher than that in the normal gastric mucosal epithelial cell line (P < 0.05). Positive TRDMT1 protein expression in the GC tissue was higher than that in the adjacent tissue. The expression of TRDMT1 was positively associated with tumor size, histological grade, invasion depth, lymph node metastasis, and tumor node metastasis (TNM) stage (P < 0.05). High TRDMT1 expression predicted poor OS of GC patients. Tumor size, differentiation degree, invasion depth, lymph node metastasis, TNM stage, and TRDMT1 expression were independent predictors of the OS of GC patients. Knockdown of TRDMT1 inhibited the migration and invasion of MKN28 cells. CONCLUSION: TRDMT1 was highly expressed in GC cell lines and tissues. TRDMT1 expression was independent predictor of the OS of GC patients. TRDMT1 knockdown reduced GC cell migration and invasion. All these results suggested that TRDMT1 has the potential to be used as a target for the diagnosis and treatment of GC.


Assuntos
Ácido Aspártico , Neoplasias Gástricas , Idoso , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Masculino , Metiltransferases , Invasividade Neoplásica , RNA de Transferência , Neoplasias Gástricas/genética
7.
Int J Mol Sci ; 22(19)2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34639202

RESUMO

The interactions of ligands with nucleic acids are central to numerous reactions in the biological cell. How such reactions are affected by harsh environmental conditions such as low temperatures, high pressures, and high concentrations of destructive ions is still largely unknown. To elucidate the ions' role in shaping habitability in extraterrestrial environments and the deep subsurface of Earth with respect to fundamental biochemical processes, we investigated the effect of selected salts (MgCl2, MgSO4, and Mg(ClO4)2) and high hydrostatic pressure (relevant for the subsurface of that planet) on the complex formation between tRNA and the ligand ThT. The results show that Mg2+ salts reduce the binding tendency of ThT to tRNA. This effect is largely due to the interaction of ThT with the salt anions, which leads to a strong decrease in the activity of the ligand. However, at mM concentrations, binding is still favored. The ions alter the thermodynamics of binding, rendering complex formation that is more entropy driven. Remarkably, the pressure favors ligand binding regardless of the type of salt. Although the binding constant is reduced, the harsh conditions in the subsurface of Earth, Mars, and icy moons do not necessarily preclude nucleic acid-ligand interactions of the type studied here.


Assuntos
Benzotiazóis/metabolismo , Cloreto de Magnésio/farmacologia , Sulfato de Magnésio/farmacologia , Percloratos/farmacologia , Pressão , RNA de Transferência/metabolismo , Temperatura , Benzotiazóis/química , Planeta Terra , Exobiologia , Meio Ambiente Extraterreno , Ligantes , Marte , Lua , RNA de Transferência/química , Termodinâmica
8.
Nat Commun ; 12(1): 5933, 2021 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-34635670

RESUMO

GTPases are regulators of cell signaling acting as molecular switches. The translational GTPase EF-G stands out, as it uses GTP hydrolysis to generate force and promote the movement of the ribosome along the mRNA. The key unresolved question is how GTP hydrolysis drives molecular movement. Here, we visualize the GTPase-powered step of ongoing translocation by time-resolved cryo-EM. EF-G in the active GDP-Pi form stabilizes the rotated conformation of ribosomal subunits and induces twisting of the sarcin-ricin loop of the 23 S rRNA. Refolding of the GTPase switch regions upon Pi release initiates a large-scale rigid-body rotation of EF-G pivoting around the sarcin-ricin loop that facilitates back rotation of the ribosomal subunits and forward swiveling of the head domain of the small subunit, ultimately driving tRNA forward movement. The findings demonstrate how a GTPase orchestrates spontaneous thermal fluctuations of a large RNA-protein complex into force-generating molecular movement.


Assuntos
Escherichia coli/genética , Fator G para Elongação de Peptídeos/química , Biossíntese de Proteínas , RNA Mensageiro/química , RNA Ribossômico 23S/química , RNA de Transferência/química , Ribossomos/metabolismo , Sítios de Ligação , Fenômenos Biomecânicos , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Hidrólise , Cinética , Modelos Moleculares , Fator G para Elongação de Peptídeos/genética , Fator G para Elongação de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/ultraestrutura , Termodinâmica
9.
Nat Commun ; 12(1): 5900, 2021 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-34625550

RESUMO

RNA polymerase III achieves high level tRNA synthesis by termination-associated reinitiation-recycling that involves the essential C11 subunit and heterodimeric C37/53. The C11-CTD (C-terminal domain) promotes Pol III active center-intrinsic RNA 3'-cleavage although deciphering function for this activity has been complicated. We show that the isolated NTD (N-terminal domain) of C11 stimulates Pol III termination by C37/53 but not reinitiation-recycling which requires the NTD-linker (NTD-L). By an approach different from what led to current belief that RNA 3'-cleavage activity is essential, we show that NTD-L can provide the essential function of Saccharomyces cerevisiae C11 whereas classic point mutations that block cleavage, interfere with active site function and are toxic to growth. Biochemical and in vivo analysis including of the C11 invariant central linker led to a model for Pol III termination-associated reinitiation-recycling. The C11 NTD and CTD stimulate termination and RNA 3'-cleavage, respectively, whereas reinitiation-recycling activity unique to Pol III requires only the NTD-linker. RNA 3'-cleavage activity increases growth rate but is nonessential.


Assuntos
RNA Polimerase III/metabolismo , Terminação da Transcrição Genética , Transcrição Genética , Domínio Catalítico , Clivagem do RNA , RNA de Transferência/biossíntese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Regiões Terminadoras Genéticas
10.
Free Radic Biol Med ; 176: 286-297, 2021 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-34624482

RESUMO

Reactive nitrogen species and nutrient deprivation are two elements of the immune response used to eliminate pathogens within phagosomes. Concomitantly, pathogenic bacteria have evolved defense systems to cope with phagosomal stressors, which include enzymes that detoxify nitric oxide (•NO) and respond to nutrient scarcity. A deeper understanding of how those defense systems are deployed under adverse conditions that contain key elements of phagosomes will facilitate targeting of those systems for therapeutic purposes. Here we investigated how Escherichia coli detoxifies •NO in the absence of useable nitrogen, because nitrogen availability is limited in phagosomes due to the removal of nitrogenous compounds (e.g., amino acids). We hypothesized that nitrogen starvation would impair •NO detoxification by E. coli because it depresses translation rates and the main E. coli defense enzyme, Hmp, is synthesized in response to •NO. However, we found that E. coli detoxifies •NO at the same rate regardless of whether useable nitrogen was present. We confirmed that the nitrogen in •NO and its autoxidation products could not be used by E. coli under our experimental conditions, and discovered that •NO eliminated differences in carbon and oxygen consumption between nitrogen-replete and nitrogen-starved cultures. Interestingly, E. coli does not consume measurable extracellular nitrogen during •NO stress despite the need to translate defense enzymes. Further, we found that RelA, which responds to uncharged tRNA, was required to observe the robustness of •NO detoxification to nitrogen starvation. These data demonstrate that E. coli is well poised to detoxify •NO in the absence of useable nitrogen and suggest that the stringent response could be a useful target to potentiate the antibacterial activity of •NO.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Óxido Nítrico , Nitrogênio , RNA de Transferência
11.
Am J Hum Genet ; 108(11): 2195-2204, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34715011

RESUMO

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.


Assuntos
Alelos , Pleiotropia Genética , Mitocôndrias/enzimologia , RNA Mitocondrial/genética , RNA de Transferência/genética , Ribonuclease P/genética , Adulto , Feminino , Humanos , Masculino , Linhagem
12.
Nat Commun ; 12(1): 5429, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34521822

RESUMO

Bacillus subtilis is a model gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins in B. subtilis, here we report broad and efficient genetic code expansion in B. subtilis by incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences between E. coli and B. subtilis stop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system in B. subtilis will help uncover an abundance of biological insights and aid genetic code expansion in other organisms.


Assuntos
Aminoácidos/genética , Aminoacil-tRNA Sintetases/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Código Genético , Aminoácidos/química , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/classificação , Aminoacil-tRNA Sintetases/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Códon , Citocinese/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma Bacteriano , Ligação Proteica , Biossíntese de Proteínas , Mapeamento de Interação de Proteínas , RNA de Transferência/genética , RNA de Transferência/metabolismo
13.
Nat Commun ; 12(1): 5513, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34535641

RESUMO

Under the Baltimore nucleic acid-based virus classification scheme, the herpesvirus human cytomegalovirus (HCMV) is a Class I virus, meaning that it contains a double-stranded DNA genome-and no RNA. Here, we report sub-particle cryoEM reconstructions of HCMV virions at 2.9 Å resolution revealing structures resembling non-coding transfer RNAs (tRNAs) associated with the virion's capsid-bound tegument protein, pp150. Through deep sequencing, we show that these RNA sequences match human tRNAs, and we built atomic models using the most abundant tRNA species. Based on our models, tRNA recruitment is mediated by the electrostatic interactions between tRNA phosphate groups and the helix-loop-helix motif of HCMV pp150. The specificity of these interactions may explain the absence of such tRNA densities in murine cytomegalovirus and other human herpesviruses.


Assuntos
Capsídeo/metabolismo , Citomegalovirus/ultraestrutura , Fosfoproteínas/metabolismo , RNA de Transferência/metabolismo , Proteínas da Matriz Viral/metabolismo , Vírion/ultraestrutura , Anticódon/metabolismo , Sequência de Bases , Linhagem Celular , Microscopia Crioeletrônica , Glutamato-tRNA Ligase/química , Glutamato-tRNA Ligase/metabolismo , Humanos , Modelos Moleculares , Fosfoproteínas/ultraestrutura , RNA Viral/ultraestrutura , Proteínas da Matriz Viral/ultraestrutura
14.
Biochemistry (Mosc) ; 86(8): 992-1002, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34488575

RESUMO

During protein synthesis, ribosome moves along mRNA to decode one codon after the other. Ribosome translocation is induced by a universally conserved protein, elongation factor G (EF-G) in bacteria and elongation factor 2 (EF-2) in eukaryotes. EF-G-induced translocation results in unwinding of the intramolecular secondary structures of mRNA by three base pairs at a time that renders the translating ribosome a processive helicase. Professor Alexander Sergeevich Spirin has made numerous seminal contributions to understanding the molecular mechanism of translocation. Here, we review Spirin's insights into the ribosomal translocation and recent advances in the field that stemmed from Spirin's pioneering work. We also discuss key remaining challenges in studies of translocase and helicase activities of the ribosome.


Assuntos
RNA Helicases/química , Ribossomos/fisiologia , Transferases/química , Transporte Biológico , Microscopia Crioeletrônica , Eucariotos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Fator 2 de Elongação de Peptídeos/química , Fator G para Elongação de Peptídeos/química , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA de Transferência/química , Ribossomos/química
16.
Methods Enzymol ; 658: 1-24, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34517943

RESUMO

Knowledge of the structural information is essential for understanding the functional details of modified RNA. Cellular non-coding RNA such as rRNA, tRNA and even viral RNAs contain a number of post-transcriptional modifications with varied degree of diversity and density. In this chapter, we discuss the use of a combination of biochemical and analytical tools such as ribonucleases and liquid chromatography coupled with mass spectrometry approaches for characterization of modified RNA. We present the protocols and alternate strategies for obtaining confident modified sequence information to facilitate the understanding of function.


Assuntos
RNA , Ribonucleases , Sequência de Bases , Cromatografia Líquida , RNA/genética , Processamento Pós-Transcricional do RNA , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribonucleases/metabolismo , Espectrometria de Massas em Tandem
17.
Methods Enzymol ; 658: 191-223, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34517947

RESUMO

Chemical modifications of RNA molecules can affect translation in multiple ways. Therefore, it is critical to understand how their absence changes cellular translation dynamics and in particular codon-specific translation. In this chapter, we discuss the application of ribosome profiling to analyze changes in codon-specific translation and differential translation in Saccharomyces cerevisiae and human cells.


Assuntos
Biossíntese de Proteínas , RNA de Transferência , Códon/genética , Códon/metabolismo , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
18.
Methods Enzymol ; 658: 225-250, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34517948

RESUMO

Transfer RNA (tRNA) is the most highly and diversely modified class of RNA in all domains of life. However, we still have only a limited understanding of the concerted action of the many enzymes that modify tRNA during tRNA maturation and the synergistic functions of tRNA modifications for protein synthesis. Here, we describe the preparation of in vitro transcribed tRNAs with a partial set of defined modifications and the use of partially modified tRNAs in biochemical assays. By comparing the affinity and activity of tRNA modification enzymes for partially modified and unmodified tRNAs, we gain insight into the preferred pathways of tRNA maturation. Additionally, partially modified tRNAs will be highly useful to investigate the importance of tRNA modifications for tRNA function during translation including the interaction with aminoacyl-tRNA synthases, translation factors and the ribosome. Thereby, the methods described here lay the foundation for understanding the mechanistic function of tRNA modifications.


Assuntos
Processamento Pós-Transcricional do RNA , RNA de Transferência , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/metabolismo
19.
Methods Enzymol ; 658: 25-47, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34517949

RESUMO

Precise and reliable mapping of modified nucleotides in RNA is a challenging task in epitranscriptomics analysis. Only deep sequencing-based methods are able to provide both, a single-nucleotide resolution and sufficient selectivity and sensitivity. A number of protocols employing specific chemical reagents to distinguish modified RNA nucleotides from canonical parental residues have already proven their performance. We developed a deep-sequencing analytical pipeline for simultaneous detection of several modified nucleotides of different nature (methylation, hydroxylation, reduction) in RNA. The AlkAniline-Seq protocol uses intrinsic fragility of the N-glycosidic bond present in certain modified residues (7-methylguanosine (m7G), 3-methylcytidine (m3C), dihydrouridine (D) and 5-hydroxycytidine (ho5C)) to induce cleavage under heat combined with alkaline conditions. The resulting RNA abasic site is decomposed by aniline-driven ß-elimination and creates a 5'-phosphate (5'-P) at the adjacent N+1 residue. This 5'-P is the crucial entry point for a highly selective ligation of sequencing adapters during the subsequent Illumina library preparation protocol. AlkAniline-Seq protocol has a very low background, and is both highly sensitive and specific. Applications of AlkAniline-Seq include mapping of m7G, m3C, D, and ho5C in variety of cellular RNAs, including in particular rRNAs and tRNAs.


Assuntos
Citidina , Guanosina , Citidina/análogos & derivados , Guanosina/análogos & derivados , Sequenciamento de Nucleotídeos em Larga Escala , RNA/genética , RNA de Transferência/genética , Análise de Sequência de RNA
20.
Methods Enzymol ; 658: 251-275, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34517950

RESUMO

The application of in vitro kinetic tools has the potential to provide important insight into the molecular mechanisms of RNA modification enzymes. Utilizing quantitative biochemical approaches can reveal information about enzyme preferences for specific substrates that are relevant for understanding modification reactions in their biological contexts. Moreover, kinetic tools have been powerfully applied to identify and characterize roles for specific amino acid residues in catalysis, which can be essential information for understanding the molecular basis for human disease, as well as for targeting these enzymes for potential therapeutic interventions. RNA methyltransferases are a particularly interesting group of RNA modification enzymes because of the diversity in structure and mechanism that has been revealed among members of this group, even including some examples of enzymes that use entirely distinct reaction mechanisms to form identical methylated nucleotides in RNA. Yet, many questions remain unanswered about how these distinct catalytic strategies are facilitated by the relevant enzyme families. We have applied in vitro kinetic analysis to specifically focus on catalytically relevant ionizations in the context of tRNA methyltransferase reactions, by measuring rates under conditions of varied pH. This analysis can be applied broadly to RNA methyltransferases to expand our understanding of these important enzymes.


Assuntos
Metiltransferases , tRNA Metiltransferases , Catálise , Humanos , Cinética , Metiltransferases/metabolismo , RNA , RNA de Transferência , Especificidade por Substrato , tRNA Metiltransferases/metabolismo
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