RESUMO
To understand how RNA dynamics is regulated and connected to its function, we investigate the folding, conformational dynamics and robustness of Xrn1 resistance of a set of flaviviral xrRNAs using SAXS, smFRET and in vitro enzymatic assays. Flaviviral xrRNAs form discrete ring-like 3D structures, in which the length of a conserved long-range pseudoknot (PK2) ranges from 2 bp to 7 bp. We find that xrRNAs' folding, conformational dynamics and Xrn1 resistance are strongly correlated and highly Mg2+-dependent, furthermore, the Mg2+-dependence is modulated by PK2 length variations. xrRNAs with long PK2 require less Mg2+ to stabilize their folding, exhibit reduced conformational dynamics and strong Xrn1 resistance even at low Mg2+, and tolerate mutations at key tertiary motifs at high Mg2+, which generally are destructive to xrRNAs with short PK2. These results demonstrate an unusual regulatory mechanism of RNA dynamics providing insights into the functions and future biomedical applications of xrRNAs.
Assuntos
Flavivirus/genética , Conformação de Ácido Nucleico , Dobramento de RNA/genética , Dobramento de RNA/fisiologiaRESUMO
Rotavirus genomes are distributed between 11 distinct RNA molecules, all of which must be selectively copackaged during virus assembly. This likely occurs through sequence-specific RNA interactions facilitated by the RNA chaperone NSP2. Here, we report that NSP2 autoregulates its chaperone activity through its C-terminal region (CTR) that promotes RNA-RNA interactions by limiting its helix-unwinding activity. Unexpectedly, structural proteomics data revealed that the CTR does not directly interact with RNA, while accelerating RNA release from NSP2. Cryo-electron microscopy reconstructions of an NSP2-RNA complex reveal a highly conserved acidic patch on the CTR, which is poised toward the bound RNA. Virus replication was abrogated by charge-disrupting mutations within the acidic patch but completely restored by charge-preserving mutations. Mechanistic similarities between NSP2 and the unrelated bacterial RNA chaperone Hfq suggest that accelerating RNA dissociation while promoting intermolecular RNA interactions may be a widespread strategy of RNA chaperone recycling.
Assuntos
Genoma Viral/genética , Dobramento de RNA/genética , RNA Viral/genética , Rotavirus/crescimento & desenvolvimento , Empacotamento do Genoma Viral/genética , Proteínas não Estruturais Virais/metabolismo , Microscopia Crioeletrônica , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Rotavirus/genética , Rotavirus/metabolismoRESUMO
MicroRNAs (miRNAs) are small regulatory RNAs involved in virtually all biological processes. Although many of them are co-expressed from clusters, little is known regarding the impact of this organization on the regulation of their accumulation. In this study, we set to decipher a regulatory mechanism controlling the expression of the ten clustered pre-miRNAs from Kaposi's sarcoma associated herpesvirus (KSHV). We measured in vitro the efficiency of cleavage of each individual pre-miRNA by the Microprocessor and found that pre-miR-K1 and -K3 were the most efficiently cleaved pre-miRNAs. A mutational analysis showed that, in addition to producing mature miRNAs, they are also important for the optimal expression of the whole set of miRNAs. We showed that this feature depends on the presence of a canonical pre-miRNA at this location since we could functionally replace pre-miR-K1 by a heterologous pre-miRNA. Further in vitro processing analysis suggests that the two stem-loops act in cis and that the cluster is cleaved in a sequential manner. Finally, we exploited this characteristic of the cluster to inhibit the expression of the whole set of miRNAs by targeting the pre-miR-K1 with LNA-based antisense oligonucleotides in cells either expressing a synthetic construct or latently infected with KSHV.
Assuntos
Regulação Viral da Expressão Gênica/genética , Herpesvirus Humano 8/genética , MicroRNAs/genética , Processamento Pós-Transcricional do RNA/genética , RNA Viral/genética , Linhagem Celular , Células HEK293 , Humanos , Oligonucleotídeos Antissenso/genética , Dobramento de RNA/genéticaRESUMO
Pre-miRNA-377 is a hairpin-shaped regulatory RNA associated with heart failure. Here, we use single-molecule optical tweezers to unzip pre-miRNA-377 and study its stability and dynamics. We show that magnesium ions have a strong stabilizing effect, and that sodium ions stabilize the hairpin more than potassium ions. The hairpin unfolds in a single step, regardless of buffer composition. Interestingly, hairpin folding occurs either in a single step (type 1) or through the formation of intermediates, in multiple steps (type 2) or gradually (type 3). Type 3 occurs only in the presence of both sodium and magnesium, while type 1 and 2 take place in all buffers, with type 1 being the most prevalent. By reducing the size of the native hairpin loop from fourteen to four nucleotides, we demonstrate that the folding heterogeneity originates from the large size of the hairpin loop. Further, while efficient pre-miRNA-377 binders are lacking, we demonstrate that the recently developed C2 ligand displays bimodal activity: it enhances the mechanical stability of the pre-miRNA-377 hairpin and perturbs its folding. The knowledge regarding pre-miRNA stability and dynamics that we provide is important in understanding its regulatory function and how it can be modulated to achieve a therapeutic effect, e.g., in heart failure treatment.
Assuntos
MicroRNAs/ultraestrutura , Dobramento de RNA/genética , Imagem Individual de Molécula/métodos , Insuficiência Cardíaca/genética , Humanos , MicroRNAs/genética , Nanotecnologia , Conformação de Ácido Nucleico , Pinças Ópticas , RNA/química , Dobramento de RNA/fisiologia , Processamento Pós-Transcricional do RNA/genética , Processamento Pós-Transcricional do RNA/fisiologiaRESUMO
Most RNA processing occurs co-transcriptionally. We interrogated nascent pol II transcripts by chemical and enzymatic probing and determined how the "nascent RNA structureome" relates to splicing, A-I editing and transcription speed. RNA folding within introns and steep structural transitions at splice sites are associated with efficient co-transcriptional splicing. A slow pol II mutant elicits extensive remodeling into more folded conformations with increased A-I editing. Introns that become more structured at their 3' splice sites get co-transcriptionally excised more efficiently. Slow pol II altered folding of intronic Alu elements where cryptic splicing and intron retention are stimulated, an outcome mimicked by UV, which decelerates transcription. Slow transcription also remodeled RNA folding around alternative exons in distinct ways that predict whether skipping or inclusion is favored, even though it occurs post-transcriptionally. Hence, co-transcriptional RNA folding modulates post-transcriptional alternative splicing. In summary, the plasticity of nascent transcripts has widespread effects on RNA processing.
Assuntos
Processamento Alternativo/genética , Processamento Pós-Transcricional do RNA/genética , RNA/genética , Transcrição Gênica/genética , Linhagem Celular , Éxons/genética , Células HEK293 , Humanos , Íntrons/genética , Dobramento de RNA/genética , RNA Polimerase II/genética , Precursores de RNA/genética , Sítios de Splice de RNA/genéticaRESUMO
Pseudoknots are important motifs for stabilizing the structure of functional RNAs. As an example, pseudoknotted hammerhead ribozymes are highly active compared to minimal ribozymes. The design of new RNA sequences that retain the function of a model RNA structure includes taking in account pseudoknots presence in the structure, which is usually a challenge for bioinformatics tools. Our method includes using "Enzymer," a software for designing RNA sequences with desired secondary structures that may include pseudoknots. Enzymer implements an efficient stochastic search and optimization algorithm to sample RNA sequences from low ensemble defect mutational landscape of an initial design template to generate an RNA sequence that is predicted to fold into the desired target structure.
Assuntos
Biologia Computacional/métodos , Desenho Assistido por Computador , Conformação de Ácido Nucleico , RNA Catalítico/química , RNA Catalítico/genética , Biologia Sintética/métodos , Algoritmos , Sequência de Bases , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Técnicas In Vitro , Cinética , Motivos de Nucleotídeos/genética , Reação em Cadeia da Polimerase/métodos , RNA/genética , Dobramento de RNA/genética , RNA Catalítico/metabolismo , Software , Transcrição GênicaRESUMO
Ribozymes are RNAs that catalyze reactions. They occur in nature, and can also be evolved in vitro to catalyze novel reactions. This chapter provides detailed protocols for using inverse folding software to design a ribozyme sequence that will fold to a known ribozyme secondary structure and for testing the catalytic activity of the sequence experimentally. This protocol is able to design sequences that include pseudoknots, which is important as all naturally occurring full-length ribozymes have pseudoknots. The starting point is the known pseudoknot-containing secondary structure of the ribozyme and knowledge of any nucleotides whose identity is required for function. The output of the protocol is a set of sequences that have been tested for function. Using this protocol, we were previously successful at designing highly active double-pseudoknotted HDV ribozymes.
Assuntos
Biologia Computacional/métodos , Vírus Delta da Hepatite/genética , Vírus Delta da Hepatite/metabolismo , RNA Catalítico/genética , RNA Catalítico/metabolismo , Sequência de Bases , Quadruplex G , Técnicas In Vitro , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , Dobramento de RNA/genética , RNA Catalítico/química , Software , Transcrição GênicaRESUMO
Kink-turns are important RNA structural modules that facilitate long-range tertiary interactions and form binding sites for members of the L7Ae family of proteins. Present in a wide variety of functional RNAs, kink-turns play key organizational roles in many RNA-based cellular processes, including translation, modification, and tRNA biogenesis. It is important to determine the contribution of kink-turns to the overall architecture of resident RNAs, as these modules dictate ribonucleoprotein (RNP) assembly and function. This chapter describes a site-directed, hydroxyl radical-mediated footprinting strategy that utilizes L7Ae-tethered chemical nucleases to experimentally validate computationally identified kink-turns in any RNA and under a wide variety of conditions. The work plan described here uses the catalytic RNase P RNA as an example to provide a blueprint for using this footprinting method to map RNA-protein interactions in other RNP complexes.
Assuntos
Proteínas Arqueais/química , Pegada de DNA/métodos , Ácido Edético/análogos & derivados , Radical Hidroxila/química , Dobramento de RNA/genética , RNA/química , Ribonuclease P/metabolismo , Sítios de Ligação , Ácido Edético/química , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , Ligação Proteica , RNA Catalítico/genética , RNA Catalítico/metabolismo , Transcrição Reversa , Ribonuclease P/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Análise de Sequência de DNARESUMO
Biochemical assays and computational analyses have discovered RNA structures throughout various transcripts. However, the roles of these structures are mostly unknown. Here we develop folded RNA element profiling with structure library (FOREST), a multiplexed affinity assay system to identify functional interactions from transcriptome-wide RNA structure datasets. We generate an RNA structure library by extracting validated or predicted RNA motifs from gene-annotated RNA regions. The RNA structure library with an affinity enrichment assay allows for the comprehensive identification of target-binding RNA sequences and structures in a high-throughput manner. As a proof-of-concept, FOREST discovers multiple RNA-protein interaction networks with quantitative scores, including translational regulatory elements that function in living cells. Moreover, FOREST reveals different binding landscapes of RNA G-quadruplex (rG4) structures-binding proteins and discovers rG4 structures in the terminal loops of precursor microRNAs. Overall, FOREST serves as a versatile platform to investigate RNA structure-function relationships on a large scale.
Assuntos
Quadruplex G , MicroRNAs/metabolismo , Biossíntese de Proteínas/genética , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Regiões 5' não Traduzidas/genética , Simulação por Computador , Conjuntos de Dados como Assunto , Ensaio de Desvio de Mobilidade Eletroforética , Fator de Iniciação 3 em Eucariotos/metabolismo , Biblioteca Gênica , Genoma Viral/genética , Células HEK293 , HIV-1/genética , Humanos , MicroRNAs/ultraestrutura , Motivos de Nucleotídeos , Estudo de Prova de Conceito , Ligação Proteica/genética , Dobramento de RNA/genética , Precursores de RNA/ultraestrutura , RNA Mensageiro/ultraestrutura , RNA Viral/metabolismo , RNA Viral/ultraestrutura , Proteínas de Ligação a RNA/metabolismoAssuntos
Processamento Pós-Transcricional do RNA/genética , RNA Viral/genética , RNA Viral/metabolismo , SARS-CoV-2/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Epigênese Genética , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Dobramento de RNA/genética , Splicing de RNA/genéticaRESUMO
RNAs begin to fold and function during transcription. Riboswitches undergo cotranscriptional switching in the context of transcription elongation, RNA folding, and ligand binding. To investigate how these processes jointly modulate the function of the folate stress-sensing Fusobacterium ulcerans ZTP riboswitch, we apply a single-molecule vectorial folding (VF) assay in which an engineered superhelicase Rep-X sequentially releases fluorescently labeled riboswitch RNA from a heteroduplex in a 5'-to-3' direction, at ~60 nt s-1 [comparable to the speed of bacterial RNA polymerase (RNAP)]. We demonstrate that the ZTP riboswitch is kinetically controlled and that its activation is favored by slower unwinding, strategic pausing between but not before key folding elements, or a weakened transcription terminator. Real-time single-molecule monitoring captures folding riboswitches in multiple states, including an intermediate responsible for delayed terminator formation. These results show how individual nascent RNAs occupy distinct channels within the folding landscape that controls the fate of the riboswitch.
Assuntos
Fusobacterium/genética , Regulação Bacteriana da Expressão Gênica , Dobramento de RNA/genética , RNA Bacteriano/genética , Riboswitch/genética , Aminoimidazol Carboxamida/metabolismo , Fusobacterium/metabolismo , Conformação de Ácido Nucleico , RNA Bacteriano/metabolismo , Ribonucleotídeos/metabolismo , Imagem Individual de Molécula , Transcrição GênicaRESUMO
Bacterial ribosomal RNAs are synthesized by a dedicated, conserved transcription-elongation complex that transcribes at high rates, shields RNA polymerase from premature termination, and supports co-transcriptional RNA folding, modification, processing, and ribosomal subunit assembly by presently unknown mechanisms. We have determined cryo-electron microscopy structures of complete Escherichia coli ribosomal RNA transcription elongation complexes, comprising RNA polymerase; DNA; RNA bearing an N-utilization-site-like anti-termination element; Nus factors A, B, E, and G; inositol mono-phosphatase SuhB; and ribosomal protein S4. Our structures and structure-informed functional analyses show that fast transcription and anti-termination involve suppression of NusA-stabilized pausing, enhancement of NusG-mediated anti-backtracking, sequestration of the NusG C-terminal domain from termination factor ρ, and the ρ blockade. Strikingly, the factors form a composite RNA chaperone around the RNA polymerase RNA-exit tunnel, which supports co-transcriptional RNA folding and annealing of distal RNA regions. Our work reveals a polymerase/chaperone machine required for biosynthesis of functional ribosomes.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Chaperonas Moleculares/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , Sítios de Ligação/genética , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestrutura , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/ultraestrutura , Biossíntese de Proteínas/genética , Dobramento de RNA/genética , RNA Ribossômico/genética , RNA Ribossômico/ultraestrutura , Proteínas Ribossômicas/ultraestrutura , Ribossomos/ultraestrutura , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/ultraestruturaRESUMO
Nucleic acid-binding proteins of the Sac10b family, also known as Alba, are widely distributed in Archaea. However, the physiological roles of these proteins have yet to be clarified. Here, we show that Sis10b, a member of the Sac10b family from the hyperthermophilic archaeon Sulfolobus islandicus, was active in RNA strand exchange, duplex RNA unwinding in vitro and RNA unfolding in a heterologous host cell. This protein exhibited temperature-dependent binding preference for ssRNA over dsRNA and was more efficient in RNA unwinding and RNA unfolding at elevated temperatures. Notably, alanine substitution of a highly conserved basic residue (K) at position 17 in Sis10b drastically reduced the ability of this protein to catalyse RNA strand exchange and RNA unwinding. Additionally, the preferential binding of Sis10b to ssRNA also depended on the presence of K17 or R17. Furthermore, normal growth was restored to a slow-growing Sis10b knockdown mutant by overproducing wild-type Sis10b but not by overproducing K17A in this mutant strain. Our results indicate that Sis10b is an RNA chaperone that likely functions most efficiently at temperatures optimal for the growth of S. islandicus, and K17 is essential for the chaperone activity of the protein.
Assuntos
Proteínas Arqueais/genética , Proteínas de Ligação a DNA/genética , Chaperonas Moleculares/genética , RNA/genética , Archaea/genética , Proteínas Arqueais/química , Proteínas de Ligação a DNA/química , Chaperonas Moleculares/química , Ligação Proteica/genética , Dobramento de RNA/genética , RNA de Cadeia Dupla/genética , Sulfolobus/química , Sulfolobus/genéticaRESUMO
DNA and RNA sequences rich in guanine can fold into noncanonical structures called G-quadruplexes (GQs), which exhibit a common stem structure of Hoogsteen hydrogen-bonded guanine tetrads and diverse loop structures. GQ sequence motifs are overrepresented in promoters, origins of replication, telomeres, and untranslated regions in mRNA, suggesting roles in modulating gene expression and preserving genomic integrity. Given these roles and unique aspects of different structures, GQs are attractive targets for drug design, but greater insight into GQ folding pathways and the interactions stabilizing them is required. Here, we performed molecular dynamics simulations to study two bimolecular GQs, a telomeric DNA GQ and the analogous telomeric repeat-containing RNA (TERRA) GQ. We applied the Drude polarizable force field, which we show outperforms the additive CHARMM36 force field in both ion retention and maintenance of the GQ folds. The polarizable simulations reveal that the GQs bind bulk K+ ions differently, and that the TERRA GQ accumulates more K+ ions, suggesting different ion interactions stabilize these structures. Nucleobase dipole moments vary as a function of position and also contribute to ion binding. Finally, we show that the TERRA GQ is more sensitive than the telomeric DNA GQ to water-mediated modulation of ion-induced dipole-dipole interactions.
Assuntos
DNA/genética , Quadruplex G , RNA/química , Telômero/genética , DNA/química , Replicação do DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Guanina/química , Humanos , Íons/química , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , RNA/genética , Dobramento de RNA/genética , Telômero/química , Telômero/classificação , Fatores de Transcrição/química , Fatores de Transcrição/genética , Água/químicaRESUMO
The signal recognition particle (SRP), responsible for co-translational protein targeting and delivery to cellular membranes, depends on the native long-hairpin fold of its RNA to confer functionality. Since RNA initiates folding during its synthesis, we used high-resolution optical tweezers to follow in real time the co-transcriptional folding of SRP RNA. Surprisingly, SRP RNA folding is robust to transcription rate changes and the presence or absence of its 5'-precursor sequence. The folding pathway also reveals the obligatory attainment of a non-native hairpin intermediate (H1) that eventually rearranges into the native fold. Furthermore, H1 provides a structural platform alternative to the native fold for RNase P to bind and mature SRP RNA co-transcriptionally. Delays in attaining the final native fold are detrimental to the cell, altogether showing that a co-transcriptional folding pathway underpins the proper biogenesis of function-essential SRP RNA.
Assuntos
Dobramento de RNA/genética , RNA/genética , Partícula de Reconhecimento de Sinal/genética , Transcrição Gênica/genética , Escherichia coli/genética , Ligação Proteica/genética , Ribossomos/genéticaRESUMO
Post-transcriptional RNA modifications, the epitranscriptome, play important roles in modulating the functions of RNA species. Modifications of rRNA are key for ribosome production and function. Identification and characterization of enzymes involved in epitranscriptome shaping is instrumental for the elucidation of the functional roles of specific RNA modifications. Ten modified sites have been thus far identified in the mammalian mitochondrial rRNA. Enzymes responsible for two of these modifications have not been characterized. Here, we identify METTL15, show that it is the main N4-methylcytidine (m4C) methyltransferase in human cells and demonstrate that it is responsible for the methylation of position C839 in mitochondrial 12S rRNA. We show that the lack of METTL15 results in a reduction of the mitochondrial de novo protein synthesis and decreased steady-state levels of protein components of the oxidative phosphorylation system. Without functional METTL15, the assembly of the mitochondrial ribosome is decreased, with the late assembly components being unable to be incorporated efficiently into the small subunit. We speculate that m4C839 is involved in the stabilization of 12S rRNA folding, therefore facilitating the assembly of the mitochondrial small ribosomal subunits. Taken together our data show that METTL15 is a novel protein necessary for efficient translation in human mitochondria.
Assuntos
Metiltransferases/genética , Mitocôndrias/genética , Ribossomos Mitocondriais/química , RNA Ribossômico/genética , Citidina/genética , Humanos , Metilação , Mitocôndrias/química , Fosforilação Oxidativa , Biossíntese de Proteínas/genética , Dobramento de RNA/genética , Processamento Pós-Transcricional do RNA/genética , RNA Ribossômico/químicaRESUMO
The IFIT (interferon-induced proteins with tetratricopeptide repeats) family constitutes a major arm of the antiviral function of type I interferon (IFN). Human IFIT1, the earliest discovered member of this family, inhibits several viruses of positivestrand RNA genome. IFIT1 specifically recognizes single-stranded RNAwith canonical 7-methylguanylate cap at the 50 end (Cap0), and inhibits their translation by competing with eIF4E (eukaryotic initiation factor 4E), an essential factor for 50Cap recognition. Recently, a novel viral mechanism of IFIT1 suppression was reported, in which an RNA hairpin in the 50 untranslated region (50UTR) of the viral genome prevented recognition by IFIT1 and enhanced virus growth. Here, I have analyzed the in silico predicted structures in the 50UTR of the genomes of the Alphaviruses, a large group of enveloped RNA virus with positive-sense single-stranded genome. The results uncovered a large ensemble of RNA secondary structures of diverse size and shape in the different viruses, which showed little correspondence to the phylogeny of the viruses. Unexpectedly, the 50UTR of several viral genomes in this family did not fold into any structure, suggesting either their extreme sensitivity to IFIT1 or the existence of alternative viral mechanisms of subverting IFIT1 function.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Alphavirus/genética , Interações Hospedeiro-Patógeno/genética , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Regiões 5' não Traduzidas , Proteínas Adaptadoras de Transdução de Sinal/química , Alphavirus/química , Animais , Genoma Viral/genética , Humanos , Imunidade Inata/genética , Metiltransferases/química , Metiltransferases/genética , Proteínas/química , Proteínas/genética , Dobramento de RNA/genética , RNA Viral/química , Proteínas de Ligação a RNA/química , UbiquitinaçãoRESUMO
3' downstream inhibitory stem plays a crucial role in locking rpoS mRNA 5' untranslated region in a self-inhibitory state. Here, we used optical tweezers to study the unfolding/refolding of rpoS inhibitory stem in the absence and presence of Mg2+. We found adding Mg2+ decreased the free energy of the RNA junction without re-arranging its secondary structure, through confirming that this RNA formed a canonical RNA three-way junction. We suspected increased free energy might change the relative orientation of different stems of rpoS and confirmed this by small angle X-ray scattering. Such changed conformation may improve Hfq-bridged annealing between sRNA and rpoS RNA inhibitory stem. We established a convenient route to analyze the changes of RNA conformation and folding dynamics by combining optical tweezers with X-ray scattering methods. This route can be easily applied in the studies of other RNA structure and ligand-RNA.
Assuntos
Regiões 3' não Traduzidas/genética , Proteínas de Bactérias/genética , Proteínas de Escherichia coli/genética , Imagem Molecular/métodos , Conformação de Ácido Nucleico , Fator sigma/genética , Regulação Bacteriana da Expressão Gênica , Magnésio/metabolismo , Pinças Ópticas , Biossíntese de Proteínas/genética , Dobramento de RNA/genética , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
In vitro synthesized RNA is used widely in studies of RNA biology, biotechnology and RNA therapeutics. However, in vitro synthesized RNA often contains impurities, such as RNAs with lengths shorter and longer than the expected runoff RNA. We have recently confirmed that longer RNA products are formed predominantly via cis self-primed extension, in which released runoff RNA folds back on itself to prime its own RNA-templated extension. In the current work, we demonstrate that addition of a DNA oligonucleotide (capture DNA) that is complementary to the 3' end of the expected runoff RNA effectively prevents self-primed extension, even under conditions commonly used for high RNA yields. Moreover, the presence of this competing capture DNA during 'high yield' transcription, leads to an increase in the yield of expected runoff RNA by suppressing the formation of undesired longer RNA byproducts.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , DNA/genética , RNA/biossíntese , Transcrição Gênica , Proteínas Virais/genética , Bacteriófago T7/genética , Sequência de Bases/genética , RNA Polimerases Dirigidas por DNA/química , Cinética , Oligonucleotídeos/genética , RNA/genética , Dobramento de RNA/genética , Moldes Genéticos , Proteínas Virais/químicaRESUMO
Synonymous mutations have been viewed as silent mutations, since they only affect the DNA and mRNA, but not the amino acid sequence of the resulting protein. Nonetheless, recent studies suggest their significant impact on splicing, RNA stability, RNA folding, translation or co-translational protein folding. Hence, we compile 659194 synonymous mutations found in human cancer and characterize their properties. We provide the user-friendly, comprehensive resource for synonymous mutations in cancer, SynMICdb ( http://SynMICdb.dkfz.de ), which also contains orthogonal information about gene annotation, recurrence, mutation loads, cancer association, conservation, alternative events, impact on mRNA structure and a SynMICdb score. Notably, synonymous and missense mutations are depleted at the 5'-end of the coding sequence as well as at the ends of internal exons independent of mutational signatures. For patient-derived synonymous mutations in the oncogene KRAS, we indicate that single point mutations can have a relevant impact on expression as well as on mRNA secondary structure.