title: Structure and interactions of the first three RNA recognition motifs of splicing factor prp24.
abstract: The essential Saccharomyces cerevisiae pre-messenger RNA splicing protein 24 (Prp24) has four RNA recognition motifs (RRMs) and facilitates U6 RNA base-pairing with U4 RNA during spliceosome assembly. Prp24 is a component of the free U6 small nuclear ribonucleoprotein particle (snRNP) but not the U4/U6 bi-snRNP, and so is thought to be displaced from U6 by U4/U6 base-pairing. The interaction partners of each of the four RRMs of Prp24 and how these interactions direct U4/U6 pairing are not known. Here we report the crystal structure of the first three RRMs and the solution structure of the first two RRMs of Prp24. Strikingly, RRM 2 forms extensive inter-domain contacts with RRMs 1 and 3. These contacts occupy much of the canonical RNA-binding faces (beta-sheets) of RRMs 1 and 2, but leave the beta-sheet of RRM 3 exposed. Previously identified substitutions in Prp24 that suppress mutations in U4 and U6 spliceosomal RNAs cluster primarily in the beta-sheet of RRM 3, but also in a conserved loop of RRM 2. RNA binding assays and chemical shift mapping indicate that a large basic patch evident on the surface of RRMs 1 and 2 is part of a high affinity U6 RNA binding site. Our results suggest that Prp24 binds free U6 RNA primarily with RRMs 1 and 2, which may remodel the U6 secondary structure. The beta-sheet of RRM 3 then influences U4/U6 pairing through interaction with an unidentified ligand.
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.
authors.forename: Euiyoung
authors.initials: E
authors.lastname: Bae
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Reiter
authors.forename: Craig A
authors.initials: CA
authors.lastname: Bingman
authors.forename: Sharon S
authors.initials: SS
authors.lastname: Kwan
authors.forename: Donghan
authors.initials: D
authors.lastname: Lee
authors.forename: George N
authors.initials: GN
authors.lastname: Phillips
authors.forename: Samuel E
authors.initials: SE
authors.lastname: Butcher
authors.forename: David A
authors.initials: DA
authors.lastname: Brow
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012326:RNA Splicing; D012331:RNA, Fungal / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000235:genetics; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: C401305:Prp24 protein, S cerevisiae; D012331:RNA, Fungal; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D017411:Ribonucleoproteins, Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C405482:U4 small nuclear RNA; C405486:U6 small nuclear RNA