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Pseudouridines in U2 snRNA stimulate the ATPase activity of Prp5 during spliceosome assembly
Author(s) -
Wu Guowei,
Adachi Hironori,
Ge Junhui,
Stephenson David,
Query Charles C,
Yu YiTao
Publication year - 2016
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.15252/embj.201593113
Subject(s) - biology , spliceosome , small nuclear rna , snrnp , genetics , atpase , prp24 , computational biology , microbiology and biotechnology , rna splicing , biochemistry , rna , enzyme , gene , rna dependent rna polymerase
Pseudouridine (Ψ) is the most abundant internal modification identified in RNA , and yet little is understood of its effects on downstream reactions. Yeast U2 sn RNA contains three conserved Ψs (Ψ35, Ψ42, and Ψ44) in the branch site recognition region ( BSRR ), which base pairs with the pre‐ mRNA branch site during splicing. Here, we show that blocks to pseudouridylation at these positions reduce the efficiency of pre‐ mRNA splicing, leading to growth‐deficient phenotypes. Restoration of pseudouridylation at these positions using designer sno RNA s results in near complete rescue of splicing and cell growth. These Ψs interact genetically with Prp5, an RNA ‐dependent ATP ase involved in monitoring the U2 BSRR ‐branch site base‐pairing interaction. Biochemical analysis indicates that Prp5 has reduced affinity for U2 sn RNA that lacks Ψ42 and Ψ44 and that Prp5 ATP ase activity is reduced when stimulated by U2 lacking Ψ42 or Ψ44 relative to wild type, resulting in inefficient spliceosome assembly. Furthermore, in vivo DMS probing analysis reveals that pseudouridylated U2, compared to U2 lacking Ψ42 and Ψ44, adopts a slightly different structure in the branch site recognition region. Taken together, our results indicate that the Ψs in U2 sn RNA contribute to pre‐ mRNA splicing by directly altering the binding/ ATP ase activity of Prp5.