A splicing-dependent transcriptional checkpoint associated with prespliceosome formation

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

Key features of the two-intron Saccharomyces cerevisiae gene SUS1 contribute to its alternative splicing

Alternative pre-mRNA splicing allows dramatic expansion of the eukaryotic proteome and facilitates cellular response to changes in environmental conditions. The Saccharomyces cerevisiae gene SUS1, which encodes a protein involved in mRNA export and histone H2B deubiquitination, contains two introns; non-canonical sequences in the first intron contribute to its retention, a common form of alternative splicing in plants and fungi. Here we show that the pattern of SUS1 splicing changes in response to environmental change such as temperature elevation, and the retained intron product is subject to nonsense-mediated decay. The activities of different splicing factors determine the pattern of SUS1 splicing, including intron retention and exon skipping. Unexpectedly, removal of the 3′ intron is affected by splicing of the upstream intron, suggesting that cross-exon interactions influence intron removal. Production of different SUS1 isoforms is important for cellular function, as we find that the temperature sensitivity and histone H2B deubiquitination defects observed in sus1Δ cells are only partially suppressed by SUS1 cDNA, but SUS1 that is able to undergo splicing complements these phenotypes. These data illustrate a role for S. cerevisiae alternative splicing in histone modification and cellular function and reveal important mechanisms for splicing of yeast genes containing multiple introns

Real-time imaging of cotranscriptional splicing reveals a kinetic model that reduces noise: implications for alternative splicing regulation

A combination of several rate-limiting steps allows for efficient control of alternative splicing

Modelling Reveals Kinetic Advantages of Co-Transcriptional Splicing

Messenger RNA splicing is an essential and complex process for the removal of intron sequences. Whereas the composition of the splicing machinery is mostly known, the kinetics of splicing, the catalytic activity of splicing factors and the interdependency of transcription, splicing and mRNA 3′ end formation are less well understood. We propose a stochastic model of splicing kinetics that explains data obtained from high-resolution kinetic analyses of transcription, splicing and 3′ end formation during induction of an intron-containing reporter gene in budding yeast. Modelling reveals co-transcriptional splicing to be the most probable and most efficient splicing pathway for the reporter transcripts, due in part to a positive feedback mechanism for co-transcriptional second step splicing. Model comparison is used to assess the alternative representations of reactions. Modelling also indicates the functional coupling of transcription and splicing, because both the rate of initiation of transcription and the probability that step one of splicing occurs co-transcriptionally are reduced, when the second step of splicing is abolished in a mutant reporter

Genome-wide studies of mRNA synthesis and degradation in eukaryotes

In recent years, the use of genome-wide technologies has revolutionized the study of eukaryotic transcription producing results for thousands of genes at every step of mRNA life. The statistical analyses of the results for a single condition, different conditions, different transcription stages, or even between different techniques, is outlining a totally new landscape of the eukaryotic transcription process. Although most studies have been conducted in the yeast Saccharomyces cerevisiae as a model cell, others have also focused on higher eukaryotes, which can also be comparatively analyzed. The picture which emerges is that transcription is a more variable process than initially suspected, with large differences between genes at each stage of the process, from initiation to mRNA degradation, but with striking similarities for functionally related genes, indicating that all steps are coordinately regulated. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing

Genome-Wide Distribution of RNA-DNA Hybrids Identifies RNase H Targets in tRNA Genes, Retrotransposons and Mitochondria

During transcription, the nascent RNA can invade the DNA template, forming extended RNA-DNA duplexes (R-loops). Here we employ ChIP-seq in strains expressing or lacking RNase H to map targets of RNase H activity throughout the budding yeast genome. In wild-type strains, R-loops were readily detected over the 35S rDNA region, transcribed by Pol I, and over the 5S rDNA, transcribed by Pol III. In strains lacking RNase H activity, R-loops were elevated over other Pol III genes, notably tRNAs, SCR1 and U6 snRNA, and were also associated with the cDNAs of endogenous TY1 retrotransposons, which showed increased rates of mobility to the 5'-flanking regions of tRNA genes. Unexpectedly, R-loops were also associated with mitochondrial genes in the absence of RNase H1, but not of RNase H2. Finally, R-loops were detected on actively transcribed protein-coding genes in the wild-type, particularly over the second exon of spliced ribosomal protein genes

Global analysis of nascent RNA reveals transcriptional pausing in terminal exons

Pre-mRNA splicing is catalyzed by the spliceosome, which can assemble on pre-mRNA cotranscriptionally. However, whether splicing generally occurs during transcription has not been addressed. Indeed, splicing catalysis is expected to occur posttranscriptionally in yeast, where the shortness of terminal exons should leave insufficient time for splicing. Here, we isolate endogenous S. cerevisiae nascent RNA and determine gene-specific splicing efficiencies and transcription profiles, using high-density tiling microarrays. Surprisingly, we find that splicing occurs cotranscriptionally for the majority of intron-containing genes. Analysis of transcription profiles reveals Pol II pausing within the terminal exons of these genes. Intronless and inefficiently spliced genes lack this pause. In silico simulations of transcription and splicing kinetics confirm that this pausing event provides sufficient time for splicing before termination. The discovery of terminal exon pausing demonstrates functional coupling of transcription and splicing near gene ends