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.