Perturbation of transcription elongation influences the fidelity of internal exon inclusion in Saccharomyces cerevisiae

Abstract

Unknown mechanisms exist to ensure that exons are not skipped during biogenesis of mRNA. Studies have connected transcription elongation with regulated alternative exon inclusion. To determine whether the relative rates of transcription elongation and spliceosome assembly might play a general role in enforcing constitutive exon inclusion, we measured exon skipping for a natural two-intron gene in which the internal exon is constitutively included in the mRNA. Mutations in this gene that subtly reduce recognition of the intron 1 branchpoint cause exon skipping, indicating that rapid recognition of the first intron is important for enforcing exon inclusion. To test the role of transcription elongation, we treated cells to increase or decrease the rate of transcription elongation. Consistent with the "first come, first served" model, we found that exon skipping in vivo is inhibited when transcription is slowed by RNAP II mutants or when cells are treated with inhibitors of elongation. Expression of the elongation factor TFIIS stimulates exon skipping, and this effect is eliminated when lac repressor is targeted to DNA encoding the second intron. A mutation in U2 snRNA promotes exon skipping, presumably because a delay in recognition of the first intron allows elongating RNA polymerase to transcribe the downstream intron. This indicates that the relative rates of elongation and splicing are tuned so that the fidelity of exon inclusion is enhanced. These findings support a general role for kinetic coordination of transcription elongation and splicing during the transcription-dependent control of splicing.

Histone H3K36 methylation regulates pre-mRNA splicing inSaccharomyces cerevisiae

“…8,10,11,21,22 Importantly, our observation is reinforced by studies from the Struhl lab that show Set2 loss has no effect on RNA Pol II elongation rate or processivity. 70 Given that Set2/ H3K36me2 is likely functioning directly in splicing, we reasoned that Set2 and/or H3K36 methylation might be important for recruiting splicing proteins to facilitate co-transcriptional spliceosome assembly 10,11 (i.e., a recruitment model).…”

“…8,21,22 Further, impaired termination has also been shown to impact proper splicing in metazoan. 78 This possibility seems less likely as a recent report revealed that deletion of SET2 had no impact on RNA Pol II elongation or processivity.…”

“…67 Given the intimate connection between transcription elongation and splicing, 8,10,11,21,22 we next wondered if the splicing defects observed in the set2D strain were a consequence of alterations in transcriptional elongation. To answer this question, we took advantage of 2 well-established transcriptional elongation defects observed in the set2D: bypass of a FACT chromatin-reorganizing complex mutant, spt16-11, 68 at semi-permissive temperature and resistance to the transcription elongation inhibitor 6-Azauracil (6-AU).…”

“…13,14 Mutations that alter transcription elongation rates or treatment with drugs that affect transcription can change alternative splicing outcomes in metazoa [15][16][17][18][19][20] and splicing efficiency in yeast. 8,21,22 For example, splicing of the DYN2 alternative splicing reporter pre-mRNA in S. cerevisiae changes when transcription elongation is slowed using the small molecules 6-Azauracil or mycophenolic acid, or by mutating the RNA Pol II subunit Rpb2. 21 A recent point mutation epistatic miniarray profile (pE-MAP) paired with genome-wide splicing microarray analysis of 53 RNA polymerase mutants in S. cerevisiae revealed that altering the rate of elongation can change the efficiency of splicing; slow elongation enhances splicing, while fast elongation reduces splicing efficiency.…”

“…8,21,22 For example, splicing of the DYN2 alternative splicing reporter pre-mRNA in S. cerevisiae changes when transcription elongation is slowed using the small molecules 6-Azauracil or mycophenolic acid, or by mutating the RNA Pol II subunit Rpb2. 21 A recent point mutation epistatic miniarray profile (pE-MAP) paired with genome-wide splicing microarray analysis of 53 RNA polymerase mutants in S. cerevisiae revealed that altering the rate of elongation can change the efficiency of splicing; slow elongation enhances splicing, while fast elongation reduces splicing efficiency. 22 Thus any protein that can alter RNA Pol II elongation rate has the potential to regulate RNA splicing.…”