From Structure to Systems: High-Resolution, Quantitative Genetic Analysis of RNA Polymerase II

Abstract

SUMMARY RNA polymerase II (RNAPII) lies at the core of dynamic control of gene expression. Using 53 RNAPII point mutants, we generated a point mutant epistatic miniarray profile (pE-MAP) comprising ~60,000 quantitative genetic interactions in Saccharomyces cerevisiae. This analysis enabled functional assignment of RNAPII subdomains and uncovered connections between individual regions and other protein complexes. Using splicing microarrays and mutants that alter elongation rates in vitro, we found an inverse relationship between RNAPII speed and in vivo splicing efficiency. Furthermore, the pE-MAP classified fast and slow mutants that favor upstream and downstream start site selection, respectively. The striking coordination of polymerization rate with transcription initiation and splicing suggests that transcription rate is tuned to regulate multiple gene expression steps. The pE-MAP approach provides a powerful strategy to understand other multifunctional machines at amino acid resolution.

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).…”

“…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.…”

“…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.…”