Hierarchical, clustered protein interactions with U4/U6 snRNA: a biochemical role for U4/U6 proteins.
During activation of the spliceosome, the U4/U6 snRNA duplex is dissociated, releasing U6 for subsequent base pairing with U2 snRNA. Proteins that directly bind the U4/U6 interaction domain potentially could mediate these structural changes. We thus investigated binding of the human U4/U6-specific proteins, 15.5K, 61K and the 20/60/90K protein complex, to U4/U6 snRNA in vitro. We demonstrate that protein 15.5K is a nucleation factor for U4/U6 snRNP assembly, mediating the interaction of 61K and 20/60/90K with U4/U6 snRNA. A similar hierarchical assembly pathway is observed for the U4atac/U6atac snRNP. In addition, we show that protein 61K directly contacts the 5' portion of U4 snRNA via a novel RNA-binding domain. Furthermore, the 20/60/90K heteromer requires stem II but not stem I of the U4/U6 duplex for binding, and this interaction involves a direct contact between protein 90K and U6. This uneven clustering of the U4/U6 snRNP-specific proteins on U4/U6 snRNA is consistent with a sequential dissociation of the U4/U6 duplex prior to spliceosome catalysis.
Nottrott
Stephanie
S
Department of Cellular Biochemistry, Max-Planck-Institute of Biophysical Chemistry, Göttingen, Germany.
Urlaub
Henning
H
Lührmann
Reinhard
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008954:Models, Biological; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D017381:Spliceosomes / Q000378:metabolism
D046911:Macromolecular Substances; D012342:RNA, Small Nuclear; D011994:Recombinant Proteins; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; C405482:U4 small nuclear RNA; C405486:U6 small nuclear RNA