RESUMO
Latent 5' splice sites, not normally used, are highly abundant in human introns, but are activated under stress and in cancer, generating thousands of nonsense mRNAs. A previously proposed mechanism to suppress latent splicing was shown to be independent of NMD, with a pivotal role for initiator-tRNA independent of protein translation. To further elucidate this mechanism, we searched for nuclear proteins directly bound to initiator-tRNA. Starting with UV-crosslinking, we identified nucleolin (NCL) interacting directly and specifically with initiator-tRNA in the nucleus, but not in the cytoplasm. Next, we show the association of ini-tRNA and NCL with pre-mRNA. We further show that recovery of suppression of latent splicing by initiator-tRNA complementation is NCL dependent. Finally, upon nucleolin knockdown we show activation of latent splicing in hundreds of coding transcripts having important cellular functions. We thus propose nucleolin, a component of the endogenous spliceosome, through its direct binding to initiator-tRNA and its effect on latent splicing, as the first protein of a nuclear quality control mechanism regulating splice site selection to protect cells from latent splicing that can generate defective mRNAs.
Assuntos
Sítios de Ligação , Fosfoproteínas/metabolismo , Sítios de Splice de RNA , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Espectrometria de Massas , Ligação Proteica , Interferência de RNA , RNA de Transferência/genética , NucleolinaRESUMO
Telomerase is a ribonucleoprotein complex that extends the 3' ends of linear chromosomes. The specialized telomerase reverse transcriptase requires a multidomain RNA (telomerase RNA, TER), which includes an integral RNA template and functionally important template-adjacent pseudoknot. The structure of the human TER pseudoknot revealed that the loops interact with the stems to form a triple helix shown to be important for activity in vitro. A similar triple helix has been predicted to form in diverse fungi TER pseudoknots. The solution NMR structure of the Kluyveromyces lactis pseudoknot, presented here, reveals that it contains a long pyrimidine motif triple helix with unexpected features that include three individual bulge nucleotides and a C(+)â¢G-C triple adjacent to a stem 2-loop 2 junction. Despite significant differences in sequence and base triples, the 3D shape of the human and K. lactis TER pseudoknots are remarkably similar. Analysis of the effects of nucleotide substitutions on cell growth and telomere lengths provides evidence that this conserved structure forms in endogenously assembled telomerase and is essential for telomerase function in vivo.
Assuntos
Kluyveromyces/enzimologia , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA/química , Telomerase/química , Sequência de Bases , Humanos , Kluyveromyces/genética , Modelos Moleculares , Mutagênese , Ressonância Magnética Nuclear Biomolecular , Pirimidinas/química , RNA/genética , Estabilidade de RNA , RNA Bacteriano/genética , RNA Fúngico/química , RNA Fúngico/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Telomerase/genéticaRESUMO
Estimating the reactivity of 2'-hydroxyl groups along an RNA chain of interest aids in the modeling of the folded RNA structure; flexible loops tend to be reactive, whereas duplex regions are generally not. Among the most useful reagents for probing 2'-hydroxyl reactivity is 1-methyl-7-nitroisatoic anhydride (1m7), but the absence of a reliable, inexpensive source has prevented widespread adoption. An existing protocol for the conversion of an inexpensive precursor 4-nitroisatoic anhydride (4NIA) recommends the use of NaH in dimethylformamide (DMF), a reagent combination that most molecular biology labs are not equipped to handle, and that does not scale safely in any case. Here we describe a safer, one-pot method for bulk conversion of 4NIA to 1m7 that reduces costs and bypasses the use of NaH. We show that 1m7 produced by this method is free of side products and can be used to probe RNA structure in vitro.
Assuntos
Oxazinas/síntese química , RNA Bacteriano/química , RNA de Transferência/química , Acilação , Escherichia coli/genética , Escherichia coli/metabolismo , Técnicas Genéticas , Química Verde , Indicadores e Reagentes/síntese química , Conformação de Ácido Nucleico , RNA Bacteriano/metabolismo , RNA de Transferência/metabolismo , Coloração e RotulagemRESUMO
Telomerase copies a short template within its integral telomerase RNA onto eukaryotic chromosome ends, compensating for incomplete replication and degradation. Telomerase action extends the proliferative potential of cells, and thus it is implicated in cancer and aging. Nontemplate regions of telomerase RNA are also crucial for telomerase function. However, they are highly divergent in sequence among species, and their roles are largely unclear. Using in silico three-dimensional modeling, constrained by mutational analysis, we propose a three-dimensional model for a pseudoknot in telomerase RNA of the budding yeast Kluyveromyces lactis. Interestingly, this structure includes a U-A.U major-groove triple helix. We confirmed the triple-helix formation in vitro using oligoribonucleotides and showed that it is essential for telomerase function in vivo. While triplex-disrupting mutations abolished telomerase function, triple compensatory mutations that formed pH-dependent G-C.C(+) triples restored the pseudoknot structure in a pH-dependent manner and partly restored telomerase function in vivo. In addition, we identified a novel type of triple helix that is formed by G-C.U triples, which also partly restored the pseudoknot structure and function. We propose that this unusual structure, so far found only in telomerase RNA, provides an essential and conserved telomerase-specific function.
Assuntos
RNA/química , RNA/metabolismo , Telomerase/química , Telomerase/metabolismo , Sequência de Bases , Dimerização , Concentração de Íons de Hidrogênio , Kluyveromyces/enzimologia , Kluyveromyces/genética , Modelos Moleculares , Mutação/genética , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Fenótipo , RNA/genética , Telomerase/genética , Telômero/genéticaRESUMO
Telomerase maintains the integrity of telomeres, the ends of linear chromosomes, by adding G-rich repeats to their 3'-ends. Telomerase RNA is an integral component of telomerase. It contains a template for the synthesis of the telomeric repeats by the telomerase reverse transcriptase. Although telomerase RNAs of different organisms are very diverse in their sequences, a functional non-template element, a pseudoknot, was predicted in all of them. Pseudoknot elements in human and the budding yeast Kluyveromyces lactis telomerase RNAs contain unusual triple-helical segments with AUU base triples, which are critical for telomerase function. Such base triples in ciliates have not been previously reported. We analyzed the pseudoknot sequences in 28 ciliate species and classified them in six different groups based on the lengths of the stems and loops composing the pseudoknot. Using miniCarlo, a helical parameter-based modeling program, we calculated 3D models for a representative of each morphological group. In all cases, the predicted structure contains at least one AUU base triple in stem 2, except for that of Colpidium colpoda, which contains unconventional GCG and AUA triples. These results suggest that base triples in a pseudoknot element are a conserved feature of all telomerases.
Assuntos
Cilióforos/genética , Modelos Moleculares , RNA de Protozoário/química , RNA/química , Telomerase/química , Animais , Sequência de Bases , Sequência Conservada , Conformação de Ácido Nucleico , RNA/classificação , RNA de Protozoário/classificação , Telomerase/classificação , Tetrahymenina/genéticaRESUMO
Pre-mRNA splicing of Pol II transcripts is executed in the mammalian cell nucleus within a huge (21 MDa) and highly dynamic RNP machine - the supraspliceosome. It is composed of four splicing active native spliceosomes, each resembling an in vitro assembled spliceosome, which are connected by the pre-mRNA. Supraspliceosomes harbor protein splicing factors and all the five-spliceosomal U snRNPs. Recent analysis of specific supraspliceosomes at defined splicing stages revealed that they harbor all five spliceosomal U snRNAs at all splicing stages. Supraspliceosomes harbor additional pre-mRNA processing components, such as the 5'-end and 3'-end processing components, and the RNA editing enzymes ADAR1 and ADAR2. The structure of the native spliceosome, at a resolution of 20 Å, was determined by cryo-EM. A unique spatial arrangement of the spliceosomal U snRNPs within the native spliceosome emerged from in-silico studies, localizing the five U snRNPs mostly within its large subunit, and sheltering the active core components deep within the spliceosomal cavity. The supraspliceosome provides a platform for coordinating the numerous processing steps that the pre-mRNA undergoes: 5' and 3'-end processing activities, RNA editing, constitutive and alternative splicing, and processing of intronic microRNAs. It also harbors a quality control mechanism termed suppression of splicing (SOS) that, under normal growth conditions, suppresses splicing at abundant intronic latent 5' splice sites in a reading frame-dependent fashion. Notably, changes in these regulatory processing activities are associated with human disease and cancer. These findings emphasize the supraspliceosome as a multi-task master regulator of pre-mRNA processing in the cell nucleus.