The In Vivo Kinetics of RNA Polymerase II Elongation during Co-Transcriptional Splicing

Abstract

Kinetic analysis shows that RNA polymerase elongation kinetics are not modulated by co-transcriptional splicing and that post-transcriptional splicing can proceed at the site of transcription without the presence of the polymerase.

Abstract

RNA processing events that take place on the transcribed pre-mRNA include capping, splicing, editing, 3′ processing, and polyadenylation. Most of these processes occur co-transcriptionally while the RNA polymerase II (Pol II) enzyme is engaged in transcriptional elongation. How Pol II elongation rates are influenced by splicing is not well understood. We generated a family of inducible gene constructs containing increasing numbers of introns and exons, which were stably integrated in human cells to serve as actively transcribing gene loci. By monitoring the association of the transcription and splicing machineries on these genes in vivo, we showed that only U1 snRNP localized to the intronless gene, consistent with a splicing-independent role for U1 snRNP in transcription. In contrast, all snRNPs accumulated on intron-containing genes, and increasing the number of introns increased the amount of spliceosome components recruited. This indicates that nascent RNA can assemble multiple spliceosomes simultaneously. Kinetic measurements of Pol II elongation in vivo, Pol II ChIP, as well as use of Spliceostatin and Meayamycin splicing inhibitors showed that polymerase elongation rates were uncoupled from ongoing splicing. This study shows that transcription elongation kinetics proceed independently of splicing at the model genes studied here. Surprisingly, retention of polyadenylated mRNA was detected at the transcription site after transcription termination. This suggests that the polymerase is released from chromatin prior to the completion of splicing, and the pre-mRNA is post-transcriptionally processed while still tethered to chromatin near the gene end.

Author Summary

The pre-mRNA emerging from RNA polymerase II during eukaryotic transcription undergoes a series of processing events. These include 5′-capping, intron excision and exon ligation during splicing, 3′-end processing, and polyadenylation. Processing events occur co-transcriptionally, meaning that a variety of enzymes assemble on the pre-mRNA while the polymerase is still engaged in transcription. The concept of co-transcriptional mRNA processing raises questions about the possible coupling between the transcribing polymerase and the processing machineries. Here we examine how the co-transcriptional assembly of the splicing machinery (the spliceosome) might affect the elongation kinetics of the RNA polymerase. Using live-cell microscopy, we followed the kinetics of transcription of genes containing increasing numbers of introns and measured the recruitment of transcription and splicing factors. Surprisingly, a sub-set of splicing factors was recruited to an intronless gene, implying that there is a polymerase-coupled scanning mechanism for intronic sequences. There was no difference in polymerase elongation rates on genes with or without introns, suggesting that the spliceosome does not modulate elongation kinetics. Experiments including inhibition of splicing or transcription, together with stochastic computational simulation, demonstrated that pre-mRNAs can be retained on the gene when polymerase termination precedes completion of splicing. Altogether we show that polymerase elongation kinetics are not affected by splicing events on the emerging pre-mRNA, that increased splicing leads to more splicing factors being recruited to the mRNA, and that post-transcriptional splicing can proceed at the site of transcription in the absence of the polymerase.