SummaryIn 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.
Prp8 protein is a highly conserved pre-mRNA splicing factor and a component of spliceosomal U5 snRNPs. Intriguingly, 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 a 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, which is disrupted by mutations that cause human RP.Nuclear pre-mRNA splicing is an essential housekeeping process in all eukaryotic cells. It is catalyzed by a large ribonucleoprotein (RNP) complex called the spliceosome, which contains the small nuclear RNPs (snRNPs) U1, U2, U4, U5 and U6, as well as many nonsnRNP proteins1, 2. Each snRNP consists of an snRNA, a set of specific proteins, and seven common Sm proteins or, in the case of U6 snRNP, seven Lsm proteins.Unexpectedly, mutations in four human snRNP-associated proteins, PRPF83, PRPF314, PRPF35 and PAP-1/RP96, 7 were found in patients with a dominantly inherited form of retinal degeneration, Retinitis pigmentosa (RP). Here, we investigate the role of Prp8p (the yeast ortholog of PRPF8) in U5 snRNP biogenesis in Saccharomyces cerevisiae, and the effect of RP mutations on this process.Biogenesis of the U snRNPs has been studied extensively in metazoans1, 8. The U1, U2, U4 and U5 snRNAs are produced as precursors in the nucleus by RNA polymerase II then exported to the cytoplasm, facilitated by nuclear cap-binding proteins and the export factors, CRM1 and PHAX8. In the cytoplasm seven Sm proteins bind to the snRNAs, facilitated by the SMN complex9, 10, and the m 7 G cap is hypermethylated to form a 2,2,7-
BackgroundRNA 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.ResultsBy 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0848-1) contains supplementary material, which is available to authorized users.
Microarray analyses were performed on yeast strains mutant for the nuclear-specific exosome components Rrp6p and Rrp47p/Lrp1p or the core component Rrp41p/Ski6p, at permissive temperature and following transfer to 37• C. 339 mRNAs showed clearly altered expression levels, with an unexpectedly high degree of heterogeneity in the different exosome mutants. In contrast, no clear alterations were seen in strains lacking the cytoplasmic exosome component Ski7p. 27 mRNAs that were overexpressed in each strain defective in the nuclear exosome are good candidates for regulation by nuclear turnover. These included the mRNA for the autoregulated RNA-binding protein Nrd1p. Northern and primer extension analyses confirmed the elevated NRD1 mRNA levels in exosome mutants, and revealed the accumulation of truncated 5 fragments of the mRNA. These contain a predicted Nrd1p-binding site, potentially sequestering the protein and disrupting its autoregulation. Several genes located immediately downstream of independently transcribed snoRNA genes were overexpressed in exosome mutants, presumably due to stabilization of the products of transcription termination read-through. Further analyses indicated that many snoRNA and snRNA genes are inefficiently terminated, but read-through transcripts into downstream ORFs are normally rapidly degraded by the exosome.
SummaryThere 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.
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 39-end cleavage and polyadenylation. This system permits pre-mRNA production, splicing, 39-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 39-end cleavage and polyadenylation, that is, cotranscriptionally.
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.
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.The excision of introns from precursor mRNAs (pre-mRNAs) occurs by two consecutive transesterification reactions in the spliceosome, a large and highly dynamic ribonucleoprotein complex (9). These chemical reactions are likely catalyzed by small nuclear RNAs (snRNAs) that exist within small nuclear ribonucleoprotein particles (snRNPs), but non-snRNP proteins also play essential roles such as conferring specificity, checking the fidelity of the process, and regulating conformational rearrangements in the spliceosome (8,34,42). Five snRNPs, called U1, U2, U4, U5, and U6, assemble on the substrate pre-mRNA to form the spliceosome. First, the U1 snRNP binds at the 5Ј splice site, followed by the U2 snRNP at the branch point, and then the U4, U5, and U6 snRNPs, in the form of a U4/U6.U5 tri-snRNP, join the assembling complex. Activation of the assembled spliceosome requires dynamic remodeling of an intricate network of RNA-RNA and RNAprotein interactions within the spliceosome such that the U1 and U4 snRNPs are released. Concomitantly, the Prp19-associated complex of proteins (nineteen complex or NTC) (27,28,35,36) associates with the spliceosome, remodeling the U5 snRNP (24, 25) and stabilizing interactions of the U5 and U6 snRNAs with the pre-mRNA (10, 11) prior to the first catalytic step of splicing.Upon completion of the splicing reaction, the spliced exon RNA (mRNA) is released and the postsplicing ribonucleoprotein complex dissociates in an active process that involves two members of the ATP-dependent DExH box RNA helicase family, Prp22 and Prp43 (3,26,32). Prp22 is needed for release of the spliced exons (32, 43), while Prp43 is required for disassembly of the spliceosome and release of the excised intron in its branched, lariat form (26). The U4 snRNP reassociates with the U6 snRNP (31, 41) to form the U4/U6 di-snRNP that will then join the U5 ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.