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.
Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Olexandr
O
Dybkov
Cindy L
CL
Will
Jochen
J
Deckert
Nastaran
N
Behzadnia
Klaus
K
Hartmuth
Reinhard
R
Lührmann
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