Abstract
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.
Highlights
Most transcripts produced by eukaryotic RNA polymerase II (RNAPII) undergo processing at their 50 ends, 30 ends, and internally
The chromatin immunoprecipitation (ChIP) method has been used to demonstrate the sequential recruitment of spliceosomal small nuclear ribonucleoprotein particles to the site of transcription, indicating spliceosome assembly on nascent transcripts in both yeast and mammalian cells (Gornemann et al, 2005; Lacadie and Rosbash, 2005; Listerman et al, 2006; Tardiff et al, 2006; Moore et al, 2006)
We propose that transcriptional pausing is imposed by a checkpoint that is associated with cotranscriptional splicing
Summary
Most transcripts produced by eukaryotic RNA polymerase II (RNAPII) undergo processing at their 50 ends (capping), 30 ends (cleavage and polyadenylation), and internally (splicing to remove introns). It is widely accepted that many of these modifications, especially capping and 30 end formation, occur cotranscriptionally, that is, before transcription is completed and the RNA is released from the site of transcription (Pandit et al, 2008; Perales and Bentley 2009). There is evidence, at least in mammalian systems, that coupling between the different processes is functionally important (Kornblihtt et al, 2004; Neugebauer, 2006; Pandit et al, 2008; Perales and Bentley, 2009)
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