Pseudouridine Affects Spliceosome Function at Multiple Points in Splicing

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry & Biophysics, 2011.Splicing is the process by which introns are removed from pre-messenger RNA. Splicing is catalyzed by the spliceosome. The spliceosome is a ribonucleoprotein comprised of small nuclear RNA (snRNA) and associated proteins. The spliceosome is dynamic, and the composition and conformation of elements which comprise the spliceosome change frequently throughout splicing. U2 snRNA is essential during splicing. U2 snRNA binds the branch region of the intron through complementary base pairing at the branch site recognition region (BSRR). The BSRR is modified to contain pseudouridine at multiple positions. These pseudouridines are important for splicing; however, their role in splicing is unknown. Saccharomyces cerevisiae contains three pseudouridines in the BSRR of U2. Addition of these pseudouridines, located at positions 35, 42 and 44 in yeast, are catalyzed by Pus7p, Snr81RNP, and Pus1p, respectively. To determine whether these pseudouridines affect splicing, genes for their respective pseudouridylases were deleted. Only the pus1Δ snR81Δ strain, which abolished pseudouridylation at positions 42 and 44, resulted in a growth defect, indicating a synergistic interaction at these positions. Additionally, new pseudouridines were introduced in the snR81Δ pus1Δ strain at positions 38 and 40 of U2. This resulted in partial suppression of the growth defect, suggesting the possibility that addition of pseudouridine at one position in the BSRR might compensate for loss of pseudouridine at another. To understand how pseudouridylation affects splicing, we examined mutant introns defective at certain steps in splicing. Opposing effects on intron mutants that affect the first and second catalytic steps resulting from the addition of pseudouridines at positions 38 and 40 in wild type U2 (U2+Ψ38,40) suggested an interaction involving Prp16p. As predicted, U2+Ψ38,40 suppressed prp16-302 cold sensitivity, indicating an interaction after the first catalytic step. In prp5 alleles, however, U2+Ψ38,40 exacerbated splicing defects; more significantly in a prp5 allele with reduced ATPase activity. Prp5p catalyzes unwinding of the branch site loop (BSL) to promote prespliceosome assembly. The BSL includes BSRR pseudouridines in its stem helix. The presence of U2+Ψ38,40 could affect BSL helix stability, altering a critical balance between competing U2 structures and influencing Prp5p activity. U2+Ψ38,40 suppression of prp16-302, however, suggests an additional interaction at a later stage, after the first step, favoring a conformation that includes U2 stem-loop IIa. In vivo structure probing of U2 supports this conclusion, indicating structural changes in U2 consistent with the genetic data. Together, these results suggest a model in which BSRR pseudouridylation affects the spliceosome at multiple steps in splicing