RESUMEN
Transfer RNAs (tRNAs) are among the most heavily modified RNA species. Posttranscriptional tRNA modifications (ptRMs) play fundamental roles in modulating tRNA structure and function and are being increasingly linked to human physiology and disease. Detection of ptRMs is often challenging, expensive, and laborious. Restriction fragment length polymorphism (RFLP) analyses study the patterns of DNA cleavage after restriction enzyme treatment and have been used for the qualitative detection of modified bases on mRNAs. It is known that some ptRMs induce specific and reproducible base "mutations" when tRNAs are reverse transcribed. For example, inosine, which derives from the deamination of adenosine, is detected as a guanosine when an inosine-containing tRNA is reverse transcribed, amplified via polymerase chain reaction (PCR), and sequenced. ptRM-dependent base changes on reverse transcription PCR amplicons generated as a consequence of the reverse transcription reaction might create or abolish endonuclease restriction sites. The suitability of RFLP for the detection and/or quantification of ptRMs has not been studied thus far. Here we show that different ptRMs can be detected at specific sites of different tRNA types by RFLP. For the examples studied, we show that this approach can reliably estimate the modification status of the sample, a feature that can be useful in the study of the regulatory role of tRNA modifications in gene expression.
Asunto(s)
Adenosina Desaminasa/metabolismo , Modelos Biológicos , Polimorfismo de Longitud del Fragmento de Restricción , Procesamiento Postranscripcional del ARN , ARN de Transferencia de Alanina/metabolismo , ARN de Transferencia de Treonina/metabolismo , Adenosina/metabolismo , Adenosina Desaminasa/química , Adenosina Desaminasa/genética , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados , Emparejamiento Base , Biología Computacional , Desaminación , Sistemas Especialistas , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Inosina/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , ARN de Transferencia de Alanina/antagonistas & inhibidores , ARN de Transferencia de Treonina/antagonistas & inhibidores , ARN de Transferencia de Valina/antagonistas & inhibidores , ARN de Transferencia de Valina/metabolismo , Transcripción Reversa , Especificidad por SustratoRESUMEN
Transfer RNAs (tRNAs) are key adaptor molecules of the genetic code that are heavily modified post-transcriptionally. Inosine at the first residue of the anticodon (position 34; I34) is an essential widespread tRNA modification that has been poorly studied thus far. The modification in eukaryotes results from a deamination reaction of adenine that is catalyzed by the heterodimeric enzyme adenosine deaminase acting on tRNA (hetADAT), composed of two subunits: ADAT2 and ADAT3. Using high-throughput small RNA sequencing (RNAseq), we show that this modification is incorporated to human tRNAs at the precursor tRNA level and during maturation. We also functionally validated the human genes encoding for hetADAT and show that the subunits of this enzyme co-localize in nucleus in an ADAT2-dependent manner. Finally, by knocking down HsADAT2, we demonstrate that variations in the cellular levels of hetADAT will result in changes in the levels of I34 modification in all its potential substrates. Altogether, we present RNAseq as a powerful tool to study post-transcriptional tRNA modifications at the precursor tRNA level and give the first insights on the biology of I34 tRNA modification in metazoans.
Asunto(s)
Inosina/metabolismo , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN de Transferencia/metabolismo , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Núcleo Celular/enzimología , Núcleo Celular/genética , Células HEK293 , Humanos , Precursores del ARN/química , ARN de Transferencia/química , Análisis de Secuencia de ARNRESUMEN
Here we present an X-ray crystallography structure of the clinically relevant tigecycline antibiotic bound to the 70S ribosome. Our structural and biochemical analysis indicate that the enhanced potency of tigecycline results from a stacking interaction with nucleobase C1054 within the decoding site of the ribosome. Single-molecule fluorescence resonance energy transfer studies reveal that, during decoding, tigecycline inhibits the initial codon recognition step of tRNA accommodation and prevents rescue by the tetracycline-resistance protein TetM.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Minociclina/análogos & derivados , Secuencia de Bases , Sitios de Unión , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Glicilglicina/química , Glicilglicina/farmacología , Minociclina/química , Minociclina/farmacología , Modelos Moleculares , Biosíntesis de Proteínas/efectos de los fármacos , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN de Transferencia/química , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Ribosomas/química , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Electricidad Estática , Relación Estructura-Actividad , Thermus thermophilus/efectos de los fármacos , Thermus thermophilus/genética , Thermus thermophilus/metabolismo , TigeciclinaRESUMEN
A mutation in the WDR4 gene, coding for a tRNA-modifying enzyme, leads to reduced levels of guanosine methylation in tRNA in patients with primordial dwarfism.
Asunto(s)
Enanismo Hipofisario/genética , Procesamiento Postranscripcional del ARN/genética , ARN de Transferencia/genética , Enanismo Hipofisario/patología , Humanos , Metilación , Mutación , ARN de Transferencia/metabolismo , Saccharomyces cerevisiaeRESUMEN
Inosine on transfer RNAs (tRNAs) are post-transcriptionally formed by a deamination mechanism of adenosines at positions 34, 37 and 57 of certain tRNAs. Despite its ubiquitous nature, the biological role of inosine in tRNAs remains poorly understood. Recent developments in the study of nucleotide modifications are beginning to indicate that the dynamics of such modifications are used in the control of specific genetic programs. Likewise, the essentiality of inosine-modified tRNAs in genome evolution and animal biology is becoming apparent. Here we review our current understanding on the role of inosine in tRNAs, the enzymes that catalyze the modification and the evolutionary link between such enzymes and other deaminases.
Asunto(s)
Adenosina/metabolismo , Evolución Molecular , Inosina/metabolismo , Edición de ARN , ARN de Transferencia/metabolismo , Adenosina Desaminasa/metabolismo , Animales , Humanos , ARN de Transferencia/químicaRESUMEN
Initiation factors guide the ribosome in the selection of mRNA and translational reading frame. We determined the kinetically favored assembly pathway of the 30S preinitiation complex (30S PIC), an early intermediate in 30S initiation complex formation in Escherichia coli. IF3 and IF2 are the first factors to arrive, forming an unstable 30S-IF2-IF3 complex. Subsequently, IF1 joins and locks the factors in a kinetically stable 30S PIC to which fMet-tRNA(fMet) is recruited. Binding of mRNA is independent of initiation factors and can take place at any time during 30S PIC assembly, depending on the cellular concentration of the mRNA and the structural determinants at the ribosome-binding site. The kinetic analysis shows both specific and cumulative effects of initiation factors as well as kinetic checkpoints of mRNA selection at the entry into translation.