RESUMEN
Nucleoside and nucleotide analogues have proven to be transformative in the treatment of viral infections and cancer. One branch of structural modification to deliver new nucleoside analogue classes explores replacement of canonical ribose oxygen with a sulfur atom. Whilst biological activity of such analogues has been shown in some cases, widespread exploration of this compound class is hitherto hampered by the lack of a straightforward and universal nucleobase diversification strategy. Herein, we present a synergistic platform enabling both biocatalytic nucleobase diversification from 4'-thiouridine in a one-pot process, and chemical functionalization to access new entities. This methodology delivers entry across pyrimidine and purine 4'-thionucleosides, paving a way for wider synthetic and biological exploration. We exemplify our approach by enzymatic synthesis of 5-iodo-4'-thiouridine on multi-milligram scale and from here switch to complete chemical synthesis of a novel nucleoside analogue probe, 5-ethynyl-4'-thiouridine. Finally, we demonstrate the utility of this probe to monitor RNA synthesis in proliferating HeLa cells, validating its capability as a new metabolic RNA labelling tool.
Asunto(s)
ARN , Tionucleósidos , Tiouridina , Tiouridina/análogos & derivados , Tiouridina/química , Tiouridina/metabolismo , Humanos , Tionucleósidos/química , Tionucleósidos/metabolismo , Tionucleósidos/síntesis química , ARN/metabolismo , ARN/química , Células HeLa , Biocatálisis , Estructura MolecularRESUMEN
Nucleoside analogues like 4-thiouridine (4sU) are used to metabolically label newly synthesized RNA. Chemical conversion of 4sU before sequencing induces T-to-C mismatches in reads sequenced from labelled RNA, allowing to obtain total and labelled RNA expression profiles from a single sequencing library. Cytotoxicity due to extended periods of labelling or high 4sU concentrations has been described, but the effects of extensive 4sU labelling on expression estimates from nucleotide conversion RNA-seq have not been studied. Here, we performed nucleotide conversion RNA-seq with escalating doses of 4sU with short-term labelling (1h) and over a progressive time course (up to 2h) in different cell lines. With high concentrations or at later time points, expression estimates were biased in an RNA half-life dependent manner. We show that bias arose by a combination of reduced mappability of reads carrying multiple conversions, and a global, unspecific underrepresentation of labelled RNA emerging during library preparation and potentially global reduction of RNA synthesis. We developed a computational tool to rescue unmappable reads, which performed favourably compared to previous read mappers, and a statistical method, which could fully remove remaining bias. All methods developed here are freely available as part of our GRAND-SLAM pipeline and grandR package.
Asunto(s)
RNA-Seq , Tiouridina , Tiouridina/metabolismo , Tiouridina/química , RNA-Seq/métodos , Humanos , ARN/genética , Análisis de Secuencia de ARN/métodos , Nucleótidos/genéticaRESUMEN
2-Thiouridine (s2U) is a nucleobase modification that confers enhanced efficiency and fidelity both on modern tRNA codon translation and on nonenzymatic and ribozyme-catalyzed RNA copying. We have discovered an unusual base pair between two 2-thiouridines that stabilizes an RNA duplex to a degree that is comparable to that of a native A:U base pair. High-resolution crystal structures indicate similar base-pairing geometry and stacking interactions in duplexes containing s2U:s2U compared to those with U:U pairs. Notably, the CâO···H-N hydrogen bond in the U:U pair is replaced with a CâS···H-N hydrogen bond in the s2U:s2U base pair. The thermodynamic stability of the s2U:s2U base pair suggested that this self-pairing might lead to an increased error frequency during nonenzymatic RNA copying. However, competition experiments show that s2U:s2U base-pairing induces only a low level of misincorporation during nonenzymatic RNA template copying because the correct A:s2U base pair outcompetes the slightly weaker s2U:s2U base pair. In addition, even if an s2U is incorrectly incorporated, the addition of the next base is greatly hindered. This strong stalling effect would further increase the effective fidelity of nonenzymatic RNA copying with s2U. Our findings suggest that s2U may enhance the rate and extent of nonenzymatic copying with only a minimal cost in fidelity.
Asunto(s)
ARN Catalítico , ARN , Tiouridina/análogos & derivados , ARN/química , Emparejamiento Base , Tiouridina/química , ARN Catalítico/química , Conformación de Ácido NucleicoRESUMEN
Quantitative comparisons of RNA levels from different samples can lead to new biological understanding if they are able to distinguish biological variation from variable sample preparation. These challenges are pronounced in comparisons that require complex biochemical manipulations (e.g. isolating polysomes to study translation). Here, we present Transcript Regulation Identified by Labeling with Nucleoside Analogues in Cell Culture (TILAC), an internally controlled approach for quantitative comparisons of RNA content. TILAC uses two metabolic labels, 4-thiouridine (s4U) and 6-thioguanosine (s6G), to differentially label RNAs in cells, allowing experimental and control samples to be pooled prior to downstream biochemical manipulations. TILAC leverages nucleoside recoding chemistry to generate characteristic sequencing signatures for each label and uses statistical modeling to compare the abundance of RNA transcripts between samples. We verified the performance of TILAC in transcriptome-scale experiments involving RNA polymerase II inhibition and heat shock. We then applied TILAC to quantify changes in mRNA association with actively translating ribosomes during sodium arsenite stress and discovered a set of transcripts that are translationally upregulated, including MCM2 and DDX5. TILAC is broadly applicable to uncover differences between samples leading to improved biological insights.
Asunto(s)
Nucleósidos , Tiouridina , Tiouridina/química , Análisis de Secuencia de ARN , ARN/química , ARN Mensajero/metabolismoRESUMEN
Both sequence enrichment and base resolution are essential for accurate sequencing analysis of low-abundance RNA. Yet they are hindered by the lack of molecular tools. Here we report a bifunctional chemical signature for RNA 4-thiouridine (4sU) enrichment sequencing with single-base resolution. This chemical signature is designed for specific 4sU labeling with two functional parts. One part is a distal alkynyl group for the biotin-assisted pulldown enrichment of target molecules via click chemistry crosslinking. The other part is a -NH group proximal to the pyrimidine ring of 4sU. It allows 4sU-to-cytosine transition during the polymerase-catalyzed extension reaction based on altering hydrogen-bonding patterns. Ultimately, the 4sU-containing RNA molecules can be enriched and accurately analyzed by single-base resolution sequencing. The proposed method also holds great potential to investigate transcriptome dynamics integrated with high-throughput sequencing.
Asunto(s)
ARN/química , Tiouridina/química , Química Clic , Citosina/química , Secuenciación de Nucleótidos de Alto Rendimiento , Espectrometría de Masas , Motivos de Nucleótidos , Pirimidinas/química , Estabilidad del ARN , Análisis de Secuencia de ARNRESUMEN
RNA structure in the influenza A virus (IAV) has been the focus of several studies that have shown connections between conserved secondary structure motifs and their biological function in the virus replication cycle. Questions have arisen on how to best recognize and understand the pandemic properties of IAV strains from an RNA perspective, but determination of the RNA secondary structure has been challenging. Herein, we used chemical mapping to determine the secondary structure of segment 8 viral RNA (vRNA) of the pandemic A/California/04/2009 (H1N1) strain of IAV. Additionally, this long, naturally occurring RNA served as a model to evaluate RNA mapping with 4-thiouridine (4sU) crosslinking. We explored 4-thiouridine as a probe of nucleotides in close proximity, through its incorporation into newly transcribed RNA and subsequent photoactivation. RNA secondary structural features both universal to type A strains and unique to the A/California/04/2009 (H1N1) strain were recognized. 4sU mapping confirmed and facilitated RNA structure prediction, according to several rules: 4sU photocross-linking forms efficiently in the double-stranded region of RNA with some flexibility, in the ends of helices, and across bulges and loops when their structural mobility is permitted. This method highlighted three-dimensional properties of segment 8 vRNA secondary structure motifs and allowed to propose several long-range three-dimensional interactions. 4sU mapping combined with chemical mapping and bioinformatic analysis could be used to enhance the RNA structure determination as well as recognition of target regions for antisense strategies or viral RNA detection.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Virus de la Influenza A/química , Gripe Humana/virología , ARN Viral/química , Tiouridina/química , Emparejamiento Base , Secuencia de Bases , Humanos , Conformación de Ácido NucleicoRESUMEN
Functionalized oligonucleotides (ONs) are widely applied as target binding molecules for biosensing and regulators for gene expression. Numerous efforts have been focused on developing facile methods for preparing these useful ONs carrying diverse modifications. Herein, we present a general method for postsynthetic modification of ONs via oxidative amination of 4-thio-2'-deoxyuridine (4SdU). 4SdU-containing ON can be derived by both alkyl and aromatic amines. Using this approach, ONs are successfully attached with alkyne/azide, biotin and dansylamide moieties, and these as-prepared ONs possess the expected biorthogonal reactivity, streptavidin affinity and fluorescent property, respectively. Furthermore, we also directly install fluorophores to the ON nucleobase based on oxidative amination of 4SdU, and these fluorophores exhibit distinct luminescence behaviors before and after conjugation. We believe our method will be a versatile strategy for constructing various functionalized ONs used in a wide range of nucleic acid applications.
Asunto(s)
Oligonucleótidos/química , Tiouridina/análogos & derivados , Aminación , Oxidación-Reducción , Tiouridina/químicaRESUMEN
Crosslinking and immunoprecipitation (CLIP) methods are powerful techniques to interrogate direct protein-RNA interactions and dissect posttranscriptional gene regulatory networks. One widely used CLIP variant is photoactivatable ribonucleoside enhanced CLIP (PAR-CLIP) that involves in vivo labeling of nascent RNAs with the photoreactive nucleosides 4-thiouridine (4SU) or 6-thioguanosine (6SG), which can efficiently crosslink to interacting proteins using UVA and UVB light. Crosslinking of 4SU or 6SG to interacting amino acids changes their base-pairing properties and results in characteristic mutations in cDNA libraries prepared for high-throughput sequencing, which can be computationally exploited to remove abundant background from non-crosslinked sequences and help pinpoint RNA binding protein binding sites at nucleotide resolution on a transcriptome-wide scale. Here we present a streamlined protocol for fluorescence-based PAR-CLIP (fPAR-CLIP) that eliminates the need to use radioactivity. It is based on direct ligation of a fluorescently labeled adapter to the 3'end of crosslinked RNA on immobilized ribonucleoproteins, followed by isolation of the adapter-ligated RNA and efficient conversion into cDNA without the previously needed size fractionation on denaturing polyacrylamide gels. These improvements cut the experimentation by half to 2 days and increases sensitivity by 10-100-fold.
Asunto(s)
ADN Complementario/genética , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Sitios de Unión , Línea Celular , Reactivos de Enlaces Cruzados/química , Electroforesis en Gel de Poliacrilamida , GTP Fosfohidrolasas/química , Biblioteca de Genes , Humanos , Inmunoprecipitación , Oligonucleótidos , Reacción en Cadena de la Polimerasa/métodos , Unión Proteica , ARN/química , Ribonucleoproteínas/genética , Sensibilidad y Especificidad , Programas Informáticos , Tiouridina/química , Rayos UltravioletaRESUMEN
Peroxidase-generated proximity labeling is in widespread use to study subcellular proteomes and the protein interaction networks in living cells, but the development of subcellular RNA labeling is limited. APEX-seq has emerged as a new method to study subcellular RNA in living cells, but the labeling of RNA still has room to improve. In this work, we describe 4-thiouridine (s4 U)-enhanced peroxidase-generated biotinylation of RNA with high efficiency. The incorporation of s4 U could introduce additional sites for RNA labeling, enhanced biotinylation was observed on monomer, model oligo RNA and total RNA. Through the s4 U metabolic approach, the inâ vivo RNA biotinylation efficiency by peroxidase catalysis was also dramatically increased, which will benefit RNA isolation and study for the spatial transcriptome.
Asunto(s)
Peroxidasa/metabolismo , ARN/metabolismo , Tiouridina/farmacología , Biotinilación , Células HEK293 , Humanos , Conformación Molecular , Tiouridina/químicaRESUMEN
Even though the mammalian mitochondrial genome (mtDNA) is very small and only codes for 13 proteins, all being subunits of the oxidative phosphorylation system, it requires several hundred nuclear encoded proteins for its maintenance and expression. These include replication and transcription factors, approximately 80 mitoribosomal proteins and many proteins involved in the posttranscriptional modification, processing, and stability of mitochondrial RNAs. In recent years, many of these factors have been identified and functionally characterized, but the complete mtRNA-interacting proteome is not firmly established. Shotgun proteomics has been used successfully to define whole-cell polyadenylated RNA (poly(A)-RNA) interacting proteomes using the nucleotide analogue 4-thiouridine (4SU) combined with UV crosslinking, poly(A)-RNA isolation and mass spectrometry to identify all poly(A)-RNA bound proteins. Although in this case also a considerable number of mitochondrial proteins were identified, the method was not specifically directed at the mitochondrial poly(A)-RNA bound proteome. Here we describe a method for enrichment of the mitochondrial poly(A)-RNA bound proteome based on 4SU labeling and UV crosslinking. The method can be applied either for isolated mitochondria prior to UV crosslinking or for whole-cell crosslinking followed by mitochondrial isolation.
Asunto(s)
Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteoma , ARN Mitocondrial/metabolismo , Proteínas de Unión al ARN/metabolismo , ADN Mitocondrial/genética , Genoma Mitocondrial , Células HEK293 , Humanos , Espectrometría de Masas , Proteínas Mitocondriales/química , Fosforilación Oxidativa , Proteómica/métodos , ARN Mensajero/química , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Tiouridina/química , Tiouridina/metabolismo , Rayos UltravioletaRESUMEN
The residue 2-thiouridine (s2U) provides a remarkable example for the "modified wobble" hypothesis, which postulates that some post-transcriptional modifications at the wobble position of tRNAs restrict recognition of degenerate codons. Through extensive molecular dynamics simulations using our χIDRP force field parameters, we demonstrate how this modification shifts the conformational ensemble from a predominantly disordered, as in the case of an RNA pentamer (GUUUC), to a substantially ordered population in Gs2UUUC. Our simulations clearly showed that the van der Waals interaction of sulfur played a major role in driving the disorder-to-order transition. The conformational redistribution and the slowing down of the transition between the clusters within the population in the presence of s2U suggest ensemble allostery to be a key mechanism that may play a general role in the functioning of the wobble modifications of tRNAs.
Asunto(s)
ARN de Transferencia/química , Tiouridina/química , Sitio Alostérico , Secuencia de Bases , Codón/química , Simulación de Dinámica Molecular , Conformación de Ácido NucleicoRESUMEN
Ribonucleic acid (RNA) is central to many life processes and, to fulfill its function, it has a substantial chemical variety in its building blocks. Enzymatic thiolation of uridine introduces 4-thiouridine (s4 U) into many bacterial transfer RNAs (tRNAs), which is used as a sensor for UV radiation. A similar modified nucleoside, 2-thiocytidine, was recently found to be sulfur-methylated especially in bacteria exposed to antibiotics and simple methylating reagents. Herein, we report the synthesis of 4-methylthiouridine (ms4 U) and confirm its presence and additional formation under stress in Escherichia coli. We used the synthetic ms4 U for isotope dilution mass spectrometry and compared its abundance to other reported tRNA damage products. In addition, we applied sophisticated stable-isotope pulse chase studies (NAIL-MS) and showed its AlkB-independent removal inâ vivo. Our findings reveal the complex nature of bacterial RNA damage repair.
Asunto(s)
Escherichia coli/metabolismo , ARN Bacteriano/metabolismo , ARN de Transferencia/metabolismo , Tiouridina/metabolismo , Modelos Moleculares , Estructura Molecular , ARN Bacteriano/química , ARN de Transferencia/química , Tiouridina/síntesis química , Tiouridina/químicaRESUMEN
Modifications found in the Anticodon Stem Loop (ASL) of tRNAs play important roles in regulating translational speed and accuracy. Threonylcarbamoyl adenosine (t6A37) and 5-methoxycarbonyl methyl-2-thiouridine (mcm5s2U34) are critical ASL modifications that have been linked to several human diseases. The model yeast Saccharomyces cerevisiae is viable despite the absence of both modifications, growth is however greatly impaired. The major observed consequence is a subsequent increase in protein aggregates and aberrant morphology. Proteomic analysis of the t6A-deficient strain (sua5 mutant) revealed a global mistranslation leading to protein aggregation without regard to physicochemical properties or t6A-dependent or biased codon usage in parent genes. However, loss of sua5 led to increased expression of soluble proteins for mitochondrial function, protein quality processing/trafficking, oxidative stress response, and energy homeostasis. These results point to a global function for t6A in protein homeostasis very similar to mcm5/s2U modifications.
Asunto(s)
Proteínas de Unión al ADN/genética , Histona Acetiltransferasas/genética , ARN de Transferencia/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Anticodón/genética , Anticodón/metabolismo , Proteínas de Unión al ADN/metabolismo , Histona Acetiltransferasas/metabolismo , Conformación de Ácido Nucleico , Fenotipo , Agregado de Proteínas/fisiología , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/fisiología , Proteínas/genética , Proteómica/métodos , ARN de Transferencia/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Termodinámica , Tiouridina/análogos & derivados , Tiouridina/químicaRESUMEN
In eukaryotic ribosomes, the conserved protein uS19, formerly known as S15, extends with its C-terminal tail to the decoding site. The cross-linking of uS19 to the A site codon has been detected using synthetic mRNAs bearing 4-thiouridine (s4U) residues. Here, we showed that the A-site tRNA prevents this cross-linking and that the P site codon does not contact uS19. Next, we focused on determining uS19-mRNA interactions in vivo by applying the photoactivatable-ribonucleoside enhancing cross-linking and immunoprecipitation method to a stable HEK293 cell line producing FLAG-tagged uS19 and grown in a medium containing s4U. We found that when translation was stopped by cycloheximide, uS19 was efficiently cross-linked to mRNA regions with a high frequency of Glu, Lys and, more rarely, Arg codons. The results indicate that the complexes, in which the A site codon is not involved in the formation of the mRNA-tRNA duplex, are present among the cycloheximide-arrested 80S complexes, which implies pausing of elongating ribosomes at the above mRNA regions. Thus, our findings demonstrate that the human ribosomal protein uS19 interacts with mRNAs during translation elongation and highlight the regions of mRNAs where ribosome pausing occurs, bringing new structural and functional insights into eukaryotic translation in vivo.
Asunto(s)
ARN Mensajero/química , Proteínas Ribosómicas/química , Ribosomas/química , Codón , Eucariontes/genética , Células HEK293 , Humanos , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , ARN de Transferencia/química , ARN de Transferencia/genética , Proteínas Ribosómicas/genética , Ribosomas/genética , Tiouridina/químicaRESUMEN
Radiosensitizing properties of substituted uridines are of great importance for radiotherapy. Very recently, we confirmed 5-iodo-4-thio-2'-deoxyuridine (ISdU) as an efficient agent, increasing the extent of tumor cell killing with ionizing radiation. To our surprise, a similar derivative of 4-thio-2'-deoxyuridine, 5-bromo-4-thio-2'-deoxyuridine (BrSdU), does not show radiosensitizing properties at all. In order to explain this remarkable difference, we carried out a radiolytic (stationary and pulse) and quantum chemical studies, which allowed the pathways to all radioproducts to be rationalized. In contrast to ISdU solutions, where radiolysis leads to 4-thio-2'-deoxyuridine and its dimer, no dissociative electron attachment (DEA) products were observed for BrSdU. This observation seems to explain the lack of radiosensitizing properties of BrSdU since the efficient formation of the uridine-5-yl radical, induced by electron attachment to the modified nucleoside, is suggested to be an indispensable attribute of radiosensitizing uridines. A larger activation barrier for DEA in BrSdU, as compared to ISdU, is probably responsible for the closure of DEA channel in the former system. Indeed, besides DEA, the XSdU anions may undergo competitive protonation, which makes the release of X- kinetically forbidden.
Asunto(s)
Halógenos/química , Fármacos Sensibilizantes a Radiaciones/química , Tiouridina/análogos & derivados , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Histonas/metabolismo , Humanos , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Fármacos Sensibilizantes a Radiaciones/farmacología , Espectrometría de Masas en Tándem , Tiouridina/química , Tiouridina/farmacologíaRESUMEN
The phenotypic adjustments of Mycobacterium tuberculosis are commonly inferred from the analysis of transcript abundance. While mechanisms of transcriptional regulation have been extensively analysed in mycobacteria, little is known about mechanisms that shape the transcriptome by regulating RNA decay rates. The aim of the present study is to identify the core components of the RNA degradosome of M. tuberculosis and to analyse their function in RNA metabolism. Using an approach involving cross-linking to 4-thiouridine-labelled RNA, we mapped the mycobacterial RNA-bound proteome and identified degradosome-related enzymes polynucleotide phosphorylase (PNPase), ATP-dependent RNA helicase (RhlE), ribonuclease E (RNase E) and ribonuclease J (RNase J) as major components. We then carried out affinity purification of eGFP-tagged recombinant constructs to identify protein-protein interactions. This identified further interactions with cold-shock proteins and novel KH-domain proteins. Engineering and transcriptional profiling of strains with a reduced level of expression of core degradosome ribonucleases provided evidence of important pleiotropic roles of the enzymes in mycobacterial RNA metabolism highlighting their potential vulnerability as drug targets.
Asunto(s)
Mycobacterium tuberculosis/metabolismo , Polirribonucleótido Nucleotidiltransferasa/metabolismo , ARN/análisis , ARN Helicasas DEAD-box/metabolismo , Endorribonucleasas/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Complejos Multienzimáticos , Mycobacterium smegmatis/metabolismo , Polirribonucleótido Nucleotidiltransferasa/genética , Proteoma , Proteómica , ARN/química , ARN Helicasas/metabolismo , Estabilidad del ARN , ARN Bacteriano/metabolismo , Ribonucleasa III/metabolismo , Ribonucleasas/metabolismo , Tiouridina/química , TranscriptomaRESUMEN
Breakthroughs in the study of the origin of life have demonstrated how some of the building blocks essential to biology could have been formed under various primordial scenarios, and could therefore have contributed to the chemical evolution of life. Missing building blocks are then sometimes inferred to be products of primitive biosynthesis, which can stretch the limits of plausibility. Here, we demonstrate the synthesis of 2'-deoxy-2-thiouridine, and subsequently 2'-deoxyadenosine and 2-deoxyribose, under prebiotic conditions. 2'-Deoxy-2-thiouridine is produced by photoreduction of 2,2'-anhydro-2-thiouridine, which is in turn formed by phosphorylation of 2-thiouridine-an intermediate of prebiotic RNA synthesis. 2'-Deoxy-2-thiouridine is an effective deoxyribosylating agent and may have functioned as such in either abiotic or proto-enzyme-catalysed pathways to DNA, as demonstrated by its conversion to 2'-deoxyadenosine by reaction with adenine, and 2-deoxyribose by hydrolysis. An alternative prebiotic phosphorylation of 2-thiouridine leads to the formation of its 5'-phosphate, showing that hypotheses in which 2-thiouridine was a key component of early RNA sequences are within the bounds of synthetic credibility.
Asunto(s)
Evolución Química , Tiouridina/análogos & derivados , Desoxiadenosinas/síntesis química , Modelos Químicos , Oxidación-Reducción , Fosforilación , Tiouridina/síntesis química , Tiouridina/química , Tiouridina/efectos de la radiación , Rayos UltravioletaRESUMEN
Many open questions in RNA biology relate to the kinetics of gene expression and the impact of RNA binding regulatory factors on processing or decay rates of particular transcripts. Steady state measurements of RNA abundance obtained from RNA-seq approaches are not able to separate the effects of transcription from those of RNA decay in the overall abundance of any given transcript, instead only giving information on the (presumed steady-state) abundances of transcripts. Through the combination of metabolic labeling and high-throughput sequencing, several groups have been able to measure both transcription rates and decay rates of the entire transcriptome of an organism in a single experiment. This review focuses on the methodology used to specifically measure RNA decay at a global level. By comparing and contrasting approaches and describing the experimental protocols in a modular manner, we intend to provide both experienced and new researchers to the field the ability to combine aspects of various protocols to fit the unique needs of biological questions not addressed by current methods.
Asunto(s)
Química Clic/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN Mensajero/metabolismo , Coloración y Etiquetado/métodos , Transcriptoma , Animales , Biotina/análogos & derivados , Biotina/química , Bromouracilo/análogos & derivados , Línea Celular , Humanos , Estabilidad del ARN , ARN Mensajero/genética , Tiouracilo/análogos & derivados , Tiouracilo/química , Tiouracilo/metabolismo , Tiouridina/química , Tiouridina/metabolismo , Uracilo/análogos & derivados , Uracilo/química , Uracilo/metabolismo , Uridina/análogos & derivados , Uridina/química , Uridina/metabolismoRESUMEN
The effect of 2'-O-(N-methylcarbamoyl)ethyl (MCE) modification on splice-switching oligonucleotides (SSO) was systematically evaluated. The incorporation of five MCE nucleotides at the 5'-termini of SSOs effectively improved the splice switching effect. In addition, the incorporation of 2'-O-(N-methylcarbamoylethyl)-5-methyl-2-thiouridine (s2TMCE), a duplex-stabilizing nucleotide with an MCE modification, into SSOs further improved splice switching. These SSOs may be useful for the treatment of genetic diseases associated with splicing errors.
Asunto(s)
Oligonucleótidos/química , Tiouridina/química , Estructura Molecular , Tiouridina/agonistas , Tiouridina/síntesis químicaRESUMEN
MicroRNAs (miRNAs) are an evolutionary conserved class of short, single-stranded noncoding RNAs (<18-22 nt in length) that act in posttranscriptional regulation of gene expression in higher eukaryotes. The abundance of a miRNA is a key feature in control of its activity and, therefore, a number of mechanisms finely regulate miRNA levels, acting at both transcriptional and posttranscriptional level. Recent evidences, including our research, highlighted the role of miRNA decay as a mechanism controlling the miRNA pool. We describe in this chapter an optimized methodology to determine miRNA degradation rates in mammalian cells. Our approach is based on metabolic pulse labeling with 4-thiouridine (4sU), a uridine analog that is incorporated in nascent RNA and allows thiol-specific biotinylation and selective pull-down of labeled RNA. In particular, given the long average half-life and the complex biogenetic process of miRNAs, we developed a "pulse-chase" protocol where 4sU is removed from the medium after a long labeling period (2-3 h pulse), and labeled RNA is purified at different time points to measure the decay of labeled molecules. By combining the 4sU-based "pulse-chase" approach with high-throughput small RNA sequencing (sRNAseq), it is possible to quantify at genome-wide level miRNA degradation rates.