U2 snRNA-protein contacts in purified human 17S U2 snRNPs and in spliceosomal A and B complexes.
The 17S U2 snRNP plays an essential role in branch point selection and catalysis during pre-mRNA splicing. Much remains to be learned about the molecular architecture of the U2 snRNP, including which proteins contact the functionally important 5' end of the U2 snRNA. Here, RNA-protein interactions within immunoaffinity-purified human 17S U2 snRNPs were analyzed by lead(II)-induced RNA cleavage and UV cross-linking. Contacts between the U2 snRNA and SF3a60, SF3b49, SF3b14a/p14 and SmG and SmB were detected. SF3b49 appears to make multiple contacts, interacting with the 5' end of U2 and nucleotides in loops I and IIb. SF3a60 also contacted different regions of the U2 snRNA, including the base of stem-loop I and a bulge in stem-loop III. Consistent with it contacting the pre-mRNA branch point adenosine, SF3b14a/p14 interacted with the U2 snRNA near the region that base pairs with the branch point sequence. A comparison of U2 cross-linking patterns obtained with 17S U2 snRNP versus purified spliceosomal A and B complexes revealed that RNA-protein interactions with stem-loop I and the branch site-interacting region of U2 are dynamic. These studies provide important insights into the molecular architecture of 17S U2 snRNPs and reveal U2 snRNP remodeling events during spliceosome assembly.
Dybkov
Olexandr
O
Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Will
Cindy L
CL
Deckert
Jochen
J
Behzadnia
Nastaran
N
Hartmuth
Klaus
K
Lührmann
Reinhard
R
D003432:Cross-Linking Reagents; D006801:Humans; D007854:Lead / Q000494:pharmacology; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012323:RNA Processing, Post-Transcriptional / Q000187:drug effects; D012342:RNA, Small Nuclear / Q000235:genetics / Q000378:metabolism*; D020038:RNA, Spliced Leader / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000528:radiation effects; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000378:metabolism*; D017381:Spliceosomes / Q000235:genetics* / Q000378:metabolism*; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D012342:RNA, Small Nuclear; D020038:RNA, Spliced Leader; D016601:RNA-Binding Proteins; D017413:Ribonucleoprotein, U2 Small Nuclear; C405484:U2 small nuclear RNA; C088049:splicing factor 3a; D007854:Lead