RESUMO
Substitution of the queuine nucleobase precursor preQ1 by an azide-containing derivative (azido-propyl-preQ1) led to incorporation of this clickable chemical entity into tRNA via transglycosylation in vitro as well as in vivo in Escherichia coli, Schizosaccharomyces pombe and human cells. The resulting semi-synthetic RNA modification, here termed Q-L1, was present in tRNAs on actively translating ribosomes, indicating functional integration into aminoacylation and recruitment to the ribosome. The azide moiety of Q-L1 facilitates analytics via click conjugation of a fluorescent dye, or of biotin for affinity purification. Combining the latter with RNAseq showed that TGT maintained its native tRNA substrate specificity in S. pombe cells. The semi-synthetic tRNA modification Q-L1 was also functional in tRNA maturation, in effectively replacing the natural queuosine in its stimulation of further modification of tRNAAsp with 5-methylcytosine at position 38 by the tRNA methyltransferase Dnmt2 in S. pombe. This is the first demonstrated in vivo integration of a synthetic moiety into an RNA modification circuit, where one RNA modification stimulates another. In summary, the scarcity of queuosinylation sites in cellular RNA, makes our synthetic q/Q system a 'minimally invasive' system for placement of a non-natural, clickable nucleobase within the total cellular RNA.
Assuntos
Nucleosídeo Q , Schizosaccharomyces , 5-Metilcitosina/metabolismo , Azidas , Biotina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Nucleosídeo Q/química , RNA de Transferência/metabolismo , RNA de Transferência de Ácido Aspártico/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , tRNA Metiltransferases/metabolismoRESUMO
During translation, messenger RNAs (mRNAs) are decoded by ribosomes which can stall for various reasons. These include chemical damage, codon composition, starvation, or translation inhibition. Trailing ribosomes can collide with stalled ribosomes, potentially leading to dysfunctional or toxic proteins. Such aberrant proteins can form aggregates and favor diseases, especially neurodegeneration. To prevent this, both eukaryotes and bacteria have evolved different pathways to remove faulty nascent peptides, mRNAs and defective ribosomes from the collided complex. In eukaryotes, ubiquitin ligases play central roles in triggering downstream responses and several complexes have been characterized that split affected ribosomes and facilitate degradation of the various components. As collided ribosomes signal translation stress to affected cells, in eukaryotes additional stress response pathways are triggered when collisions are sensed. These pathways inhibit translation and modulate cell survival and immune responses. Here, we summarize the current state of knowledge about rescue and stress response pathways triggered by ribosome collisions.
Assuntos
Biossíntese de Proteínas , Ribossomos , Ubiquitinação , Ribossomos/genética , Ribossomos/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
A distinctive feature of prokaryotic gene expression is the absence of 5'-capped RNA. In eukaryotes, 5',5'-triphosphate-linked 7-methylguanosine protects messenger RNA from degradation and modulates maturation, localization and translation. Recently, the cofactor nicotinamide adenine dinucleotide (NAD) was reported as a covalent modification of bacterial RNA. Given the central role of NAD in redox biochemistry, posttranslational protein modification and signalling, its attachment to RNA indicates that there are unknown functions of RNA in these processes and undiscovered pathways in RNA metabolism and regulation. The unknown identity of NAD-modified RNAs has so far precluded functional analyses. Here we identify NAD-linked RNAs from bacteria by chemo-enzymatic capture and next-generation sequencing (NAD captureSeq). Among those identified, specific regulatory small RNAs (sRNAs) and sRNA-like 5'-terminal fragments of certain mRNAs are particularly abundant. Analogous to a eukaryotic cap, 5'-NAD modification is shown in vitro to stabilize RNA against 5'-processing by the RNA-pyrophosphohydrolase RppH and against endonucleolytic cleavage by ribonuclease (RNase) E. The nudix phosphohydrolase NudC decaps NAD-RNA and thereby triggers RNase-E-mediated RNA decay, while being inactive against triphosphate-RNA. In vivo, â¼13% of the abundant sRNA RNAI is NAD-capped in the presence, and â¼26% in the absence, of functional NudC. To our knowledge, this is the first description of a cap-like structure and a decapping machinery in bacteria.
Assuntos
Escherichia coli/genética , NAD/metabolismo , Capuzes de RNA/química , Capuzes de RNA/metabolismo , RNA Bacteriano/química , RNA Bacteriano/isolamento & purificação , Hidrolases Anidrido Ácido/metabolismo , Endorribonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Pirofosfatases/metabolismo , Capuzes de RNA/isolamento & purificação , Estabilidade de RNA , RNA Bacteriano/metabolismo , Análise de SequênciaRESUMO
Here, we present a simple, modular and efficient strategy that allows the 3'-terminal labeling of DNA, regardless of whether it has been chemically or enzymatically synthesized or isolated from natural sources. We first incorporate a range of modified nucleotides at the 3'-terminus, using terminal deoxynucleotidyl transferase. In the second step, we convert the incorporated nucleotides, using either of four highly efficient click chemistry-type reactions, namely copper-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, Staudinger ligation or Diels-Alder reaction with inverse electron demand. Moreover, we create internal modifications, making use of either ligation or primer extension, after the nucleotidyl transferase step, prior to the click reaction. We further study the influence of linker variants on the reactivity of azides in different click reactions. We find that different click reactions exhibit distinct substrate preferences, a fact that is often overlooked, but should be considered when labeling oligonucleotides or other biomolecules with click chemistry. Finally, our findings allowed us to extend our previously published RNA labeling strategy to the use of a different copper-free click chemistry, namely the Staudinger ligation.
Assuntos
Química Click , DNA Nucleotidilexotransferase , DNA/química , Azidas/química , DNA/metabolismo , Nucleotídeos/metabolismoRESUMO
The combination of Reverse Transcription (RT) and high-throughput sequencing has emerged as a powerful combination to detect modified nucleotides in RNA via analysis of either abortive RT-products or of the incorporation of mismatched dNTPs into cDNA. Here we simultaneously analyze both parameters in detail with respect to the occurrence of N-1-methyladenosine (m(1)A) in the template RNA. This naturally occurring modification is associated with structural effects, but it is also known as a mediator of antibiotic resistance in ribosomal RNA. In structural probing experiments with dimethylsulfate, m(1)A is routinely detected by RT-arrest. A specifically developed RNA-Seq protocol was tailored to the simultaneous analysis of RT-arrest and misincorporation patterns. By application to a variety of native and synthetic RNA preparations, we found a characteristic signature of m(1)A, which, in addition to an arrest rate, features misincorporation as a significant component. Detailed analysis suggests that the signature depends on RNA structure and on the nature of the nucleotide 3' of m(1)A in the template RNA, meaning it is sequence dependent. The RT-signature of m(1)A was used for inspection and confirmation of suspected modification sites and resulted in the identification of hitherto unknown m(1)A residues in trypanosomal tRNA.
Assuntos
Adenosina/análogos & derivados , Sequenciamento de Nucleotídeos em Larga Escala , RNA/química , Transcrição Reversa , Análise de Sequência de RNA , Adenosina/análise , Animais , Humanos , Aprendizado de Máquina , Camundongos , Homologia de Sequência do Ácido NucleicoRESUMO
Catalytic RNAs are attractive objects for studying molecular evolution. To understand how RNA libraries can evolve from randomness toward highly active catalysts, we analyze the original samples that led to the discovery of Diels-Alderase ribozymes by next-generation sequencing. Known structure-activity relationships are used to correlate abundance with catalytic performance. We find that efficient catalysts arose not just from selection for reactivity among the members of the starting library, but from improvement of less potent precursors by mutations. We observe changes in the ribozyme population in response to increasing selection pressure. Surprisingly, even after many rounds of enrichment, the libraries are highly diverse, suggesting that potential catalysts are more abundant in random space than generally thought. To highlight the use of next-generation sequencing as a tool for in vitro selections, we also apply this technique to a recent, less characterized ribozyme selection. Making use of the correlation between sequence evolution and catalytic activity, we predict mutations that improve ribozyme activity and validate them biochemically. Our study reveals principles underlying ribozyme in vitro selections and provides guidelines to render future selections more efficient, as well as to predict the conservation of key structural elements, allowing the rational improvement of catalysts.
Assuntos
RNA Catalítico/química , Evolução Molecular Direcionada , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNARESUMO
The modification of RNA with fluorophores, affinity tags and reactive moieties is of enormous utility for studying RNA localization, structure and dynamics as well as diverse biological phenomena involving RNA as an interacting partner. Here we report a labeling approach in which the RNA of interest--of either synthetic or biological origin--is modified at its 3'-end by a poly(A) polymerase with an azido-derivatized nucleotide. The azide is later on conjugated via copper-catalyzed or strain-promoted azide-alkyne click reaction. Under optimized conditions, a single modified nucleotide of choice (A, C, G, U) containing an azide at the 2'-position can be incorporated site-specifically. We have identified ligases that tolerate the presence of a 2'-azido group at the ligation site. This azide is subsequently reacted with a fluorophore alkyne. With this stepwise approach, we are able to achieve site-specific, internal backbone-labeling of de novo synthesized RNA molecules.
Assuntos
Química Click/métodos , Cobre/química , Polinucleotídeo Adenililtransferase/metabolismo , RNA/química , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Azidas/química , Carboidratos/química , Catálise , Escherichia coli/genética , Corantes Fluorescentes , Nucleotídeos/química , Nucleotídeos/metabolismo , Nucleotidiltransferases/metabolismo , RNA/metabolismo , Leveduras/enzimologiaRESUMO
Ribosome-associated quality control (RQC) pathways monitor and respond to ribosome stalling. Using in vivo UV-crosslinking and mass spectrometry, we identified a C-terminal region in Hel2/Rqt1 as an RNA binding domain. Complementary crosslinking and sequencing data for Hel2 revealed binding to 18S rRNA and translated mRNAs. Hel2 preferentially bound mRNAs upstream and downstream of the stop codon. C-terminal truncation of Hel2 abolished the major 18S crosslink and polysome association, and altered mRNA binding. HEL2 deletion caused loss of RQC and, we report here, no-go decay (NGD), with comparable effects for Hel2 truncation including the RNA-binding site. Asc1 acts upstream of Hel2 in RQC and asc1∆ impaired Hel2 binding to 18S and mRNA. In conclusion: Hel2 is recruited or stabilized on translating 40S ribosomal subunits by interactions with 18S rRNA and Asc1. This 18S interaction is required for Hel2 function in RQC and NGD. Hel2 probably interacts with mRNA during translation termination.
Assuntos
RNA Ribossômico 18S/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Mutação/genética , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
We report site-specific triple click labeling for DNA and RNA in a one-pot setup by performing inverse electron demand Diels-Alder reaction and strain-promoted and copper catalyzed click reactions sequentially. Our methodology can be applied to long and short nucleic acids alike, and could have broader potential for labeling other (bio-)molecules.
Assuntos
Química Click/métodos , Reação de Cicloadição/métodos , DNA/química , Corantes Fluorescentes/química , RNA/química , Coloração e Rotulagem/métodos , Alcinos/síntese química , Alcinos/química , Azidas/síntese química , Azidas/química , Catálise , Cobre/química , DNA/síntese química , Corantes Fluorescentes/síntese química , RNA/síntese químicaRESUMO
Here we describe a protocol for NAD captureSeq that allows for the identification of nicotinamide-adenine dinucleotide (NAD)-capped RNA sequences in total RNA samples from different organisms. NAD-capped RNA is first chemo-enzymatically biotinylated with high efficiency, permitting selective capture on streptavidin beads. Then, a highly efficient library preparation protocol tailored to immobilized, 5'-modified RNA is applied, with adaptor ligation to the RNA's 3' terminus and reverse transcription (RT) performed on-bead. Then, cDNA is released into solution, tailed, ligated to a second adaptor and PCR-amplified. After next-generation sequencing (NGS) of the DNA library, enriched sequences are identified by comparison with a control sample in which the first step of chemo-enzymatic biotinylation is omitted. Because the downstream protocol does not necessarily rely on NAD-modified but on 'clickable' or biotin-modified RNA, it can be applied to other RNA modifications or RNA-biomolecule interactions. The central part of this protocol can be completed in â¼7 d, excluding preparatory steps, sequencing and bioinformatic analysis.