title: Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly.
abstract: Base-pairing of U4 and U6 snRNAs during di-snRNP assembly requires large-scale remodeling of RNA structure that is chaperoned by the U6 snRNP protein Prp24. We investigated the mechanism of U4/U6 annealing in vitro using an assay that enables visualization of ribonucleoprotein complexes and faithfully recapitulates known in vivo determinants for the process. We find that annealing, but not U6 RNA binding, is highly dependent on the electropositive character of a 20 Å-wide groove on the surface of Prp24. During annealing, we observe the formation of a stable ternary complex between U4 and U6 RNAs and Prp24, indicating that displacement of Prp24 in vivo requires additional factors. Mutations that stabilize the U6 'telestem' helix increase annealing rates by up to 15-fold, suggesting that telestem formation is rate-limiting for U4/U6 pairing. The Lsm2-8 complex, which binds adjacent to the telestem at the 3' end of U6, provides a comparable rate enhancement. Collectively, these data identify domains of the U6 snRNP that are critical for one of the first steps in assembly of the megaDalton U4/U6.U5 tri-snRNP complex, and lead to a dynamic model for U4/U6 pairing that involves a striking degree of evolved cooperativity between protein and RNA.
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.
authors.forename: Allison L
authors.initials: AL
authors.lastname: Didychuk
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI 53706, USA.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Montemayor
authors.affiliation: Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI 53706, USA dabrow@wisc.edu.
authors.forename: David A
authors.initials: DA
authors.lastname: Brow
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA butcher@biochem.wisc.edu.
authors.forename: Samuel E
authors.initials: SE
authors.lastname: Butcher
mesh_terms: D001483:Base Sequence; D001667:Binding, Competitive; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000235:genetics / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C401305:Prp24 protein, S cerevisiae; D012331:RNA, Fungal; D012342:RNA, Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C405482:U4 small nuclear RNA; C405486:U6 small nuclear RNA