RESUMO
There is good evidence for functional interactions between splicing and transcription in eukaryotes, but how and why these processes are coupled remain unknown. Prp5 protein (Prp5p) is an RNA-stimulated adenosine triphosphatase (ATPase) required for prespliceosome formation in yeast. We demonstrate through in vivo RNA labeling that, in addition to a splicing defect, the prp5-1 mutation causes a defect in the transcription of intron-containing genes. We present chromatin immunoprecipitation evidence for a transcriptional elongation defect in which RNA polymerase that is phosphorylated at Ser5 of the largest subunit's heptad repeat accumulates over introns and that this defect requires Cus2 protein. A similar accumulation of polymerase was observed when prespliceosome formation was blocked by a mutation in U2 snRNA. These results indicate the existence of a transcriptional elongation checkpoint that is associated with prespliceosome formation during cotranscriptional spliceosome assembly. We propose a role for Cus2p as a potential checkpoint factor in transcription.
Assuntos
RNA Nuclear Pequeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Spliceossomos/metabolismo , Transcrição Gênica , Trifosfato de Adenosina/metabolismo , Imunoprecipitação da Cromatina , RNA Helicases DEAD-box/metabolismo , Éxons , Genes Fúngicos , Íntrons , Mutação , Fosforilação , RNA Polimerase II/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/genéticaRESUMO
The auxin-inducible degron (AID) is a useful technique to rapidly deplete proteins of interest in nonplant eukaryotes. Depletion is achieved by addition of the plant hormone auxin to the cell culture, which allows the auxin-binding receptor, TIR1, to target the AID-tagged protein for degradation by the proteasome. Fast depletion of the target protein requires good expression of TIR1 protein, but as we show here, high levels of TIR1 may cause uncontrolled depletion of the target protein in the absence of auxin. To enable conditional expression of TIR1 to a high level when required, we regulated the expression of TIR1 using the ß-estradiol expression system. This is a fast-acting gene induction system that does not cause secondary effects on yeast cell metabolism. We demonstrate that combining the AID and ß-estradiol systems results in a tightly controlled and fast auxin-induced depletion of nuclear target proteins. Moreover, we show that depletion rate can be tuned by modulating the duration of ß-estradiol preincubation. We conclude that TIR1 protein is a rate-limiting factor for target protein depletion in yeast, and we provide new tools that allow tightly controlled, tuneable, and efficient depletion of essential proteins whereas minimising secondary effects.
Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Choque Térmico/genética , Ácidos Indolacéticos/metabolismo , Proteínas Nucleares/genética , Saccharomycetales/genética , Estradiol , Expressão Gênica , Transporte Proteico , Proteólise , Saccharomycetales/metabolismo , Ativação TranscricionalRESUMO
Pre-messenger RNA splicing involves multi-step assembly of the large spliceosome complexes that catalyse the two consecutive trans-esterification reactions, resulting in intron removal. There is evidence that proof-reading mechanisms monitor the fidelity of this complex process. Transcripts that fail these fidelity tests are thought to be directed to degradation pathways, permitting the splicing factors to be recycled. While studying the roles of splicing factors in vivo, in budding yeast, we performed targeted depletion of individual proteins, and analysed the effect on co-transcriptional spliceosome assembly and splicing efficiency. Unexpectedly, depleting factors such as Prp16 or Prp22, that are known to function at the second catalytic step or later in the splicing pathway, resulted in a defect in the first step of splicing, and accumulation of arrested spliceosomes. Through a kinetic analysis of newly synthesized RNA, we observed that a second step splicing defect (the primary defect) was rapidly followed by the first step of splicing defect. Our results show that knocking down a splicing factor can quickly lead to a recycling defect with splicing factors sequestered in stalled complexes, thereby limiting new rounds of splicing. We demonstrate that this 'feed-back' effect can be minimized by depleting the target protein more gradually or only partially, allowing a better separation between primary and secondary effects. Our findings indicate that splicing surveillance mechanisms may not always cope with spliceosome assembly defects, and suggest that work involving knock-down of splicing factors or components of other large complexes should be carefully monitored to avoid potentially misleading conclusions.
Assuntos
Retroalimentação Fisiológica/efeitos dos fármacos , Splicing de RNA/efeitos dos fármacos , RNA Fúngico/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Spliceossomos/efeitos dos fármacos , Adenosina Trifosfatases/deficiência , Adenosina Trifosfatases/genética , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Ácidos Indolacéticos/farmacologia , Clivagem do RNA/efeitos dos fármacos , RNA Helicases/deficiência , RNA Helicases/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/deficiência , Fatores de Processamento de RNA/genética , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/genética , Spliceossomos/metabolismoRESUMO
In eukaryotic cells, there is evidence for functional coupling between transcription and processing of pre-mRNAs. To better understand this coupling, we performed a high-resolution kinetic analysis of transcription and splicing in budding yeast. This revealed that shortly after induction of transcription, RNA polymerase accumulates transiently around the 3' end of the intron on two reporter genes. This apparent transcriptional pause coincides with splicing factor recruitment and with the first detection of spliced mRNA and is repeated periodically thereafter. Pausing requires productive splicing, as it is lost upon mutation of the intron and restored by suppressing the splicing defect. The carboxy-terminal domain of the paused polymerase large subunit is hyperphosphorylated on serine 5, and phosphorylation of serine 2 is first detected here. Phosphorylated polymerase also accumulates around the 3' splice sites of constitutively expressed, endogenous yeast genes. We propose that transcriptional pausing is imposed by a checkpoint associated with cotranscriptional splicing.
Assuntos
RNA Polimerase II/metabolismo , Splicing de RNA/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Genes Fúngicos/genética , Genes Reporter/genética , Íntrons/genética , Fosforilação , Estrutura Terciária de Proteína , RNA Polimerase II/química , Sítios de Splice de RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição GênicaRESUMO
Pre-mRNA splicing involves two transesterification steps catalyzed by the spliceosome. How RNA substrates are positioned in each step and the molecular rearrangements involved, remain obscure. Here, we show that mutations in PRP16, PRP8, SNU114 and the U5 snRNA that affect this process interact genetically with CWC21, that encodes the yeast orthologue of the human SR protein, SRm300/SRRM2. Our microarray analysis shows changes in 3' splice site selection at elevated temperature in a subset of introns in cwc21Δ cells. Considering all the available data, we propose a role for Cwc21p positioning the 3' splice site at the transition to the second step conformation of the spliceosome, mediated through its interactions with the U5 snRNP. This suggests a mechanism whereby SRm300/SRRM2, might influence splice site selection in human cells.
Assuntos
Proteínas de Transporte/metabolismo , Sítios de Splice de RNA , Proteínas de Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Deleção de Genes , Genes Fúngicos , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , Precursores de RNA/química , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , Fatores de Processamento de RNA , RNA Fúngico/química , RNA Fúngico/genética , RNA Fúngico/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/química , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5/química , Ribonucleoproteína Nuclear Pequena U5/genética , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/química , Spliceossomos/genéticaRESUMO
RNA helicases are essential for virtually all cellular processes, however, their regulation is poorly understood. The activities of eight RNA helicases are required for pre-mRNA splicing. Amongst these, Brr2p is unusual in having two helicase modules, of which only the amino-terminal helicase domain appears to be catalytically active. Using genetic and biochemical approaches, we investigated interaction of the carboxy-terminal helicase module, in particular the carboxy-terminal Sec63-2 domain, with the splicing RNA helicase Prp16p. Combining mutations in BRR2 and PRP16 suppresses or enhances physical interaction and growth defects in an allele-specific manner, signifying functional interactions. Notably, we show that Brr2p Sec63-2 domain can modulate the ATPase activity of Prp16p in vitro by interfering with its ability to bind RNA. We therefore propose that the carboxy-terminal helicase module of Brr2p acquired a regulatory function that allows Brr2p to modulate the ATPase activity of Prp16p in the spliceosome by controlling access to its RNA substrate/cofactor.
Assuntos
Adenosina Trifosfatases/metabolismo , RNA Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Alelos , RNA Helicases DEAD-box/metabolismo , Temperatura Alta , Íntrons , Mutação , Estrutura Terciária de Proteína , RNA/metabolismo , RNA Helicases/química , RNA Helicases/genética , Splicing de RNA , Fatores de Processamento de RNA , Ribonucleoproteínas Nucleares Pequenas/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Pre-ribosomal particles undergo numerous structural changes during maturation, but their high complexity and short lifetimes make these changes very difficult to follow in vivo. In consequence, pre-ribosome structure and composition have largely been inferred from purified particles and analyzed in vitro. Here we describe techniques for kinetic analyses of the changes in pre-ribosome structure in living cells of Saccharomyces cerevisiae. To allow this, in vivo structure probing by DMS modification was combined with affinity purification of newly synthesized 20S pre-rRNA over a time course of metabolic labeling with 4-thiouracil. To demonstrate that this approach is generally applicable, we initially analyzed the accessibility of the region surrounding cleavage site D site at the 3' end of the mature 18S rRNA region of the pre-rRNA. This revealed a remarkably flexible structure throughout 40S subunit biogenesis, with little stable RNA-protein interaction apparent. Analysis of folding in the region of the 18S central pseudoknot was consistent with previous data showing U3 snoRNA-18S rRNA interactions. Dynamic changes in the structure of the hinge between helix 28 (H28) and H44 of pre-18S rRNA were consistent with recently reported interactions with the 3' guide region of U3 snoRNA. Finally, analysis of the H18 region indicates that the RNA structure matures early, but additional protection appears subsequently, presumably reflecting protein binding. The structural analyses described here were performed on total, affinity-purified, newly synthesized RNA, so many classes of RNA and RNA-protein complex are potentially amenable to this approach.
Assuntos
Ribossomos/química , Ribossomos/metabolismo , Sequência de Bases , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA , RNA Fúngico/química , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Ribossômico/química , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Ribossômico 18S/química , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ésteres do Ácido SulfúricoRESUMO
We describe methods for obtaining a quantitative description of RNA processing at high resolution in budding yeast. As a model gene expression system, we constructed tetON (for induction studies) and tetOFF (for repression, derepression, and RNA degradation studies) yeast strains with a series of reporter genes integrated in the genome under the control of a tetO7 promoter. Reverse transcription and quantitative real-time-PCR (RT-qPCR) methods were adapted to allow the determination of mRNA abundance as the average number of copies per cell in a population. Fluorescence in situ hybridization (FISH) measurements of transcript numbers in individual cells validated the RT-qPCR approach for the average copy-number determination despite the broad distribution of transcript levels within a population of cells. In addition, RT-qPCR was used to distinguish the products of the different steps in splicing of the reporter transcripts, and methods were developed to map and quantify 3'-end cleavage and polyadenylation. This system permits pre-mRNA production, splicing, 3'-end maturation and degradation to be quantitatively monitored with unprecedented kinetic detail, suitable for mathematical modeling. Using this approach, we demonstrate that reporter transcripts are spliced prior to their 3'-end cleavage and polyadenylation, that is, cotranscriptionally.
Assuntos
Genes Reporter , Processamento de Terminações 3' de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Saccharomyces cerevisiae , Algoritmos , Estudos de Avaliação como Assunto , Processamento de Imagem Assistida por Computador , Hibridização in Situ Fluorescente/métodos , Cinética , Modelos Biológicos , Modelos Genéticos , Processamento de Terminações 3' de RNA/fisiologia , Precursores de RNA/análise , Precursores de RNA/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
In Saccharomyces cerevisiae, Cwc21p is a protein of unknown function that is associated with the NineTeen Complex (NTC), a group of proteins involved in activating the spliceosome to promote the pre-mRNA splicing reaction. Here, we show that Cwc21p binds directly to two key splicing factors-namely, Prp8p and Snu114p-and becomes the first NTC-related protein known to dock directly to U5 snRNP proteins. Using a combination of proteomic techniques we show that the N-terminus of Prp8p contains an intramolecular fold that is a Snu114p and Cwc21p interacting domain (SCwid). Cwc21p also binds directly to the C-terminus of Snu114p. Complementary chemical cross-linking experiments reveal reciprocal protein footprints between the interacting Prp8 and Cwc21 proteins, identifying the conserved cwf21 domain in Cwc21p as a Prp8p binding site. Genetic and functional interactions between Cwc21p and Isy1p indicate that they have related functions at or prior to the first catalytic step of splicing, and suggest that Cwc21p functions at the catalytic center of the spliceosome, possibly in response to environmental or metabolic changes. We demonstrate that SRm300, the only SR-related protein known to be at the core of human catalytic spliceosomes, is a functional ortholog of Cwc21p, also interacting directly with Prp8p and Snu114p. Thus, the function of Cwc21p is likely conserved from yeast to humans.
Assuntos
Biocatálise , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/química , Sequência Conservada , Humanos , Dados de Sequência Molecular , Ligação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5/genética , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Alinhamento de SequênciaRESUMO
The U5 snRNA loop 1 aligns the 5' and 3' exons for ligation during the second step of pre-mRNA splicing. U5 is intimately associated with Prp8, which mediates pre-mRNA repositioning within the catalytic core of the spliceosome and interacts directly with U5 loop 1. The genome-wide effect of three U5 loop 1 mutants has been assessed by microarray analysis. These mutants exhibited impaired and improved splicing of subsets of pre-mRNAs compared to wild-type U5. Analysis of pre-mRNAs that accumulate revealed a change in base prevalence at specific positions near the splice sites. Analysis of processed pre-mRNAs exhibiting mRNA accumulation revealed a bias in base prevalence at one position within the 5' exon. While U5 loop 1 can interact with some of these positions the base bias is not directly related to sequence changes in loop 1. All positions that display a bias in base prevalence are at or next to positions known to interact with Prp8. Analysis of Prp8 in the presence of the three U5 loop 1 mutants revealed that the most severe mutant displayed reduced Prp8 stability. Depletion of U5 snRNA in vivo also resulted in reduced Prp8 stability. Our data suggest that certain mutations in U5 loop 1 perturb the stability of Prp8 and may affect interactions of Prp8 with a subset of pre-mRNAs influencing their splicing. Therefore, the integrity of U5 is important for the stability of Prp8 during splicing and provides one possible explanation for why U5 loop 1 and Prp8 are so highly conserved.
Assuntos
Processamento Alternativo , Mutação/genética , Precursores de RNA/genética , RNA Nuclear Pequeno/genética , Ribonucleoproteína Nuclear Pequena U4-U6/química , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5/química , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Bases , Genoma Fúngico , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U5/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/fisiologiaRESUMO
The Ntr1 and Ntr2 proteins of Saccharomyces cerevisiae have been reported to interact with proteins involved in pre-mRNA splicing, but their roles in the splicing process are unknown. We show here that they associate with a postsplicing complex containing the excised intron and the spliceosomal U2, U5, and U6 snRNAs, supporting a link with a late stage in the pre-mRNA splicing process. Extract from cells that had been metabolically depleted of Ntr1 has low splicing activity and accumulates the excised intron. Also, the level of U4/U6 di-snRNP is increased but those of the free U5 and U6 snRNPs are decreased in Ntr1-depleted extract, and increased levels of U2 and decreased levels of U4 are found associated with the U5 snRNP protein Prp8. These results suggest a requirement for Ntr1 for turnover of the excised intron complex and recycling of snRNPs. Ntr1 interacts directly or indirectly with the intron release factor Prp43 and is required for its association with the excised intron. We propose that Ntr1 promotes release of excised introns from splicing complexes by acting as a spliceosome receptor or RNA-targeting factor for Prp43, possibly assisted by the Ntr2 protein.
Assuntos
RNA Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , RNA Helicases DEAD-box , Íntrons/genética , Ligação Proteica , Splicing de RNA/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
As evidenced from mammalian cells the eukaryotic translation initiation factor eIF4G has a putative role in nuclear RNA metabolism. Here we investigate whether this role is conserved in the yeast Saccharomyces cerevisiae. Using a combination of in vitro and in vivo methods, we show that, similar to mammalian eIF4G, yeast eIF4G homologues, Tif4631p and Tif4632p, are present both in the nucleus and the cytoplasm. We show that both eIF4G proteins interact efficiently in vitro with UsnRNP components of the splicing machinery. More specifically, Tif4631p and Tif4632p interact efficiently with U1 snRNA in vitro. In addition, Tif4631p and Tif4632p associate with protein components of the splicing machinery, namely Snu71p and Prp11p. To further delineate these interactions, we map the regions of Tif4631p and Tif4632p that are important for the interaction with Prp11p and Snu71p and we show that addition of these regions to splicing reactions in vitro has a dominant inhibitory effect. The observed interactions implicate eIF4G in aspects of pre-mRNA processing. In support of this hypothesis, deletion of one of the eIF4G isoforms results in accumulation of un-spliced precursors for a number of endogenous genes, in vivo. In conclusion these observations are suggestive of the involvement of yeast eIF4G in pre-mRNA metabolism.
Assuntos
Fator de Iniciação 4F em Eucariotos/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Nuclear Pequeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fator de Iniciação Eucariótico 4G , Spliceossomos/metabolismoRESUMO
The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of the RNA produced during a brief period of labelling. This is done by attaching a biotin moiety to the incorporated thio group and affinity purifying, using streptavidin coated beads. Achieving a good yield of pure, newly synthesized RNA that is free of pre-existing RNA makes shorter labelling times possible and permits increased temporal resolution in kinetic studies. This is a protocol for very specific, high yield purification of newly synthesized RNA. The protocol presented here describes how RNA is extracted from the yeast Saccharomyces cerevisiae. However, the protocol for purification of thiolated RNA from total RNA should be effective using RNA from any organism once it has been extracted from the cells. The purified RNA is suitable for analysis by many widely used techniques, such as reverse transcriptase-qPCR, RNA-seq and SLAM-seq. The specificity, sensitivity and flexibility of this technique allow unparalleled insights into RNA metabolism.
Assuntos
RNA/química , RNA/metabolismo , Tiouracila/análogos & derivados , Biotina/metabolismo , Cromatografia de Afinidade , Regulação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Cinética , RNA/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae , Estreptavidina , Tiouracila/químicaRESUMO
The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting them for degradation. This system is exploited in many non-plant eukaryotes, such that a target protein, tagged with the AID motif, is degraded upon auxin addition. The level of TIR1 expression is critical; excessive expression leads to degradation of the AID-tagged protein even in the absence of auxin, whereas low expression leads to slow depletion. A ß-estradiol-inducible AID system was created, with expression of TIR1 under the control of a ß-estradiol inducible promoter. The level of TIR1 is tunable by changing the time of incubation with ß-estradiol before auxin addition. This protocol describes how to rapidly deplete a target protein using the AID system. The appropriate ß-estradiol incubation time depends on the abundance of the target protein. Therefore, efficient depletion depends on optimal timing that also minimizes auxin-independent depletion.
Assuntos
Proteínas de Plantas/metabolismo , Proteólise , Receptores de Superfície Celular/metabolismo , Estradiol , Ácidos Indolacéticos/metabolismo , Transporte ProteicoRESUMO
Genomes are promiscuously transcribed, necessitating mechanisms that facilitate the sorting of RNA for function or destruction. The polyA (pA) tail is one such distinguishing feature, which in the Saccharomyces cerevisiae nucleus is bound by the Nab2p protein, yielding transcript protection. As Nab2p also contacts the main nuclear export factor Mex67p, we asked whether transport kinetics contributes to RNA sorting. Indeed, 3' end sequencing of newly transcribed pA+ RNAs demonstrates that nuclear depletion of Mex67p elicits their instant and global decay. A similar phenotype is evident upon inactivation of other export factors and proportional to the amount of nuclear pA+ RNA. As RNA expression is partially rescued by Nab2p overexpression, we propose that an export block out-titrates Nab2p onto nuclear-retained pA+ RNA, reducing the pool of Nab2p available to protect new transcripts. More generally, we suggest that nuclear RNA decay, negotiated by Nab2p availability, aids in balancing cellular transcript supply with demand.
Assuntos
Núcleo Celular/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , HumanosRESUMO
Transcriptomics, the analysis of the complement of mRNAs transcribed from a cell's genome, currently focuses mainly on mature, processed mRNAs. However, posttranscriptional processing of primary transcripts can significantly affect both the quantity and the structure of the mature mRNAs and therefore of the protein products. Recently, the development of an intron-specific microarray has permitted a preliminary analysis of the splicing of all intron-containing transcripts in Saccharomyces cerevisiae. Here, we discuss the findings and what might be learned from this kind of approach.
Assuntos
Íntrons , Splicing de RNA , Proteínas Fúngicas/genética , Precursores de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Precursores de RNA , Processamento Pós-Transcricional do RNA , RNA Mensageiro , Saccharomyces cerevisiae , Transcrição GênicaRESUMO
BACKGROUND: RNA levels detected at steady state are the consequence of multiple dynamic processes within the cell. In addition to synthesis and decay, transcripts undergo processing. Metabolic tagging with a nucleotide analog is one way of determining the relative contributions of synthesis, decay and conversion processes globally. RESULTS: By improving 4-thiouracil labeling of RNA in Saccharomyces cerevisiae we were able to isolate RNA produced during as little as 1 minute, allowing the detection of nascent pervasive transcription. Nascent RNA labeled for 1.5, 2.5 or 5 minutes was isolated and analyzed by reverse transcriptase-quantitative polymerase chain reaction and RNA sequencing. High kinetic resolution enabled detection and analysis of short-lived non-coding RNAs as well as intron-containing pre-mRNAs in wild-type yeast. From these data we measured the relative stability of pre-mRNA species with different high turnover rates and investigated potential correlations with sequence features. CONCLUSIONS: Our analysis of non-coding RNAs reveals a highly significant association between non-coding RNA stability, transcript length and predicted secondary structure. Our quantitative analysis of the kinetics of pre-mRNA splicing in yeast reveals that ribosomal protein transcripts are more efficiently spliced if they contain intron secondary structures that are predicted to be less stable. These data, in combination with previous results, indicate that there is an optimal range of stability of intron secondary structures that allows for rapid splicing.
Assuntos
Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismo , Transcriptoma , Íntrons , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA não Traduzido/química , RNA não Traduzido/genética , Saccharomyces cerevisiae/genética , Tiouracila/análogos & derivados , Tiouracila/químicaRESUMO
Prp8 protein (Prp8p) is a highly conserved pre-mRNA splicing factor and a component of spliceosomal U5 small nuclear ribonucleoproteins (snRNPs). Although it is ubiquitously expressed, mutations in the C terminus of human Prp8p cause the retina-specific disease retinitis pigmentosa (RP). The biogenesis of U5 snRNPs is poorly characterized. We present evidence for a cytoplasmic precursor U5 snRNP in yeast that lacks the mature U5 snRNP component Brr2p and depends on a nuclear localization signal in Prp8p for its efficient nuclear import. The association of Brr2p with the U5 snRNP occurs within the nucleus. RP mutations in Prp8p in yeast result in nuclear accumulation of the precursor U5 snRNP, apparently as a consequence of disrupting the interaction of Prp8p with Brr2p. We therefore propose a novel assembly pathway for U5 snRNP complexes that is disrupted by mutations that cause human RP.