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Molecular and Cellular Biology, April 2006, p. 2803-2816, Vol. 26, No. 7
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.7.2803-2816.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

U2 snRNA-Protein Contacts in Purified Human 17S U2 snRNPs and in Spliceosomal A and B Complexes

Olexandr Dybkov, Cindy L. Will, Jochen Deckert, Nastaran Behzadnia, Klaus Hartmuth, and Reinhard Lührmann*

Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany

Received 28 November 2005/ Returned for modification 23 December 2005/ Accepted 13 January 2006

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.

* Corresponding author. Mailing address: Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany. Phone: 49-551-2011407. Fax: 49-551-2011197. E-mail: Reinhard.Luehrmann{at}mpi-bpc.mpg.de.

Molecular and Cellular Biology, April 2006, p. 2803-2816, Vol. 26, No. 7
0022-538X/06/$08.00+0     doi:10.1128/MCB.26.7.2803-2816.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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