title: Splicing proofreading at 5' splice sites by ATPase Prp28p.
abstract: Fidelity and efficiency of pre-mRNA splicing are critical for generating functional mRNAs, but how such accuracy in 5' splice site (SS) selection is attained is not fully clear. Through a series of yeast genetic screens, we isolated alleles of prp28 that improve splicing of suboptimal 5'SS substrates, demonstrating that WT-Prp28p proofreads, and consequently rejects, poor 5'SS. Prp28p is thought to facilitate the disruption of 5'SS-U1 snRNA pairing to allow for 5'SS-U6 snRNA pairing in the catalytic spliceosome; unexpectedly, 5'SS proofreading by Prp28p is dependent on competition with the stability of the 5'SS:U6 duplex, but not the 5'SS:U1 duplex. E404K, the strongest prp28 allele containing a mutation located in the linker region between adenosine triphosphatase (ATPase) subdomains, exhibited lower RNA-binding activity and enhanced splicing of suboptimal substrates before first-step catalysis, suggesting that decreased Prp28p activity allows longer time for suboptimal 5'SS substrates to pair with U6 snRNA and thereby reduces splicing fidelity. Residue E404 is critical for providing high splicing activity, demonstrated here in both yeast and Drosophila cells. Thus, the subdomain linker in Prp28p plays important roles both in splicing efficiency across species and in proofreading of 5'SS.
authors: Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors: Fei
authors: F
authors: Yang
authors: Xiu-Ye
authors: XY
authors: Wang
authors: Zhi-Min
authors: ZM
authors: Zhang
authors: Jia
authors: J
authors: Pu
authors: Yu-Jie
authors: YJ
authors: Fan
authors: Jiahai
authors: J
authors: Zhou
authors: Charles C
authors: CC
authors: Query
authors: Yong-Zhen
authors: YZ
authors: Xu
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000483:Alleles; D000818:Animals; D002460:Cell Line; D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004330:Drosophila / Q000235:genetics; D009154:Mutation; D022821:RNA Splice Sites; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D022821:RNA Splice Sites; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; C405483:U1 small nuclear RNA; C405486:U6 small nuclear RNA; D000251:Adenosine Triphosphatases; C088939:PRP28 protein, S cerevisiae; D053487:DEAD-box RNA Helicases