The U11/U12 snRNP 65K protein acts as a molecular bridge, binding the U12 snRNA and U11-59K protein.
U11 and U12 interact cooperatively with the 5' splice site and branch site of pre-mRNA as a stable preformed di-snRNP complex, thereby bridging the 5' and 3' ends of the intron within the U12-dependent prespliceosome. To identify proteins contributing to di-snRNP formation and intron bridging, we investigated protein-protein and protein-RNA interactions between components of the U11/U12 snRNP. We demonstrate that the U11/U12-65K protein possesses dual binding activity, interacting directly with U12 snRNA via its C-terminal RRM and the U11-associated 59K protein via its N-terminal half. We provide evidence that, in contrast to the previously published U12 snRNA secondary structure model, the 3' half of U12 forms an extended stem-loop with a highly conserved seven-nucleotide loop and that the latter serves as the 65K binding site. Addition of an oligonucleotide comprising the 65K binding site to an in vitro splicing reaction inhibited U12-dependent, but not U2-dependent, pre-mRNA splicing. Taken together, these data suggest that U11/U12-65K and U11-59K contribute to di-snRNP formation and intron bridging in the minor prespliceosome.
Benecke
Heike
H
Department of Cellular Biochemistry, MPI of Biophysical Chemistry, Göttingen, Germany.
Lührmann
Reinhard
R
Will
Cindy L
CL
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D024202:Electrophoretic Mobility Shift Assay; D019143:Evolution, Molecular; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012342:RNA, Small Nuclear / Q000235:genetics*; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D017381:Spliceosomes / Q000235:genetics / Q000378:metabolism
D012342:RNA, Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; C098762:U11 small nuclear ribonucleoprotein, human; C552126:U11-U12 small nuclear ribonucleoprotein; C096986:U12 small nuclear RNA