title: SF3B1/Hsh155 HEAT motif mutations affect interaction with the spliceosomal ATPase Prp5, resulting in altered branch site selectivity in pre-mRNA splicing.
abstract: Mutations in the U2 snRNP component SF3B1 are prominent in myelodysplastic syndromes (MDSs) and other cancers and have been shown recently to alter branch site (BS) or 3' splice site selection in splicing. However, the molecular mechanism of altered splicing is not known. We show here that hsh155 mutant alleles in Saccharomyces cerevisiae, counterparts of SF3B1 mutations frequently found in cancers, specifically change splicing of suboptimal BS pre-mRNA substrates. We found that Hsh155p interacts directly with Prp5p, the first ATPase that acts during spliceosome assembly, and localized the interacting regions to HEAT (Huntingtin, EF3, PP2A, and TOR1) motifs in SF3B1 associated with disease mutations. Furthermore, we show that mutations in these motifs from both human disease and yeast genetic screens alter the physical interaction with Prp5p, alter branch region specification, and phenocopy mutations in Prp5p. These and other data demonstrate that mutations in Hsh155p and Prp5p alter splicing because they change the direct physical interaction between Hsh155p and Prp5p. This altered physical interaction results in altered loading (i.e., "fidelity") of the BS-U2 duplex into the SF3B complex during prespliceosome formation. These results provide a mechanistic framework to explain the consequences of intron recognition and splicing of SF3B1 mutations found in disease.
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032 China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Tang
authors.affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461 USA.
authors.forename: Susana
authors.initials: S
authors.lastname: Rodriguez-Santiago
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032 China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032 China.
authors.forename: Jia
authors.initials: J
authors.lastname: Pu
authors.affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461 USA.
authors.forename: Andrea
authors.initials: A
authors.lastname: Yuste
authors.affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461 USA.
authors.forename: Varun
authors.initials: V
authors.lastname: Gupta
authors.affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461 USA.
authors.forename: Alberto
authors.initials: A
authors.lastname: Moldón
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032 China.
authors.forename: Yong-Zhen
authors.initials: YZ
authors.lastname: Xu
authors.affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461 USA.
authors.forename: Charles C
authors.initials: CC
authors.lastname: Query
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007438:Introns / Q000235:genetics; D009154:Mutation; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics; D000072260:RNA Splicing Factors / Q000235:genetics* / Q000378:metabolism*; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017381:Spliceosomes / Q000235:genetics
chemical_list: C405527:HSH155 protein, S cerevisiae; D010750:Phosphoproteins; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D017413:Ribonucleoprotein, U2 Small Nuclear; C112491:SF3B1 protein, human; D029701:Saccharomyces cerevisiae Proteins; C064447:PRP5 protein, S cerevisiae; D053487:DEAD-box RNA Helicases
keywords: HEAT motif; Prp5; SF3B1/Hsh155; disease mutation; pre-mRNA splicing fidelity