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Molecular and Cellular Biology, May 2008, p. 3548-3560, Vol. 28, No. 10
0270-7306/08/$08.00+0     doi:10.1128/MCB.01928-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The U11-48K Protein Contacts the 5' Splice Site of U12-Type Introns and the U11-59K Protein ^,

Janne J. Turunen,^1, Cindy L. Will,^2, Michael Grote,² Reinhard Lührmann,² and Mikko J. Frilander^1*

Institute of Biotechnology, PL 56 Viikinkaari 9, 00014 University of Helsinki, Helsinki, Finland,¹ MPI of Biophysical Chemistry, Department of Cellular Biochemistry, Am Fassberg 11, 37077 Göttingen, Germany²

Received 25 October 2007/ Returned for modification 11 December 2007/ Accepted 7 March 2008

Little is currently known about proteins that make contact with the pre-mRNA in the U12-dependent spliceosome and thereby contribute to intron recognition. Using site-specific cross-linking, we detected an interaction between the U11-48K protein and U12-type 5' splice sites (5'ss). This interaction did not require branch point recognition and was sensitive to 5'ss mutations, suggesting that 48K interacts with the 5'ss during the first steps of prespliceosome assembly in a sequence-dependent manner. RNA interference-induced knockdown of 48K in HeLa cells led to reduced cell growth and the inhibition of U12-type splicing, as well as the activation of cryptic, U2-type splice sites, suggesting that 48K plays a critical role in U12-type intron recognition. 48K knockdown also led to reduced levels of U11/U12 di-snRNP, indicating that 48K contributes to the stability and/or formation of this complex. In addition to making contact with the 5'ss, 48K interacts with the U11-59K protein, a protein at the interface of the U11/U12 di-snRNP. These studies provide important insights into the protein-mediated recognition of the U12-type 5'ss, as well as functionally important interactions within the U11/U12 di-snRNP.

Published ahead of print on 17 March 2008.

Supplemental material for this article may be found at http://mcb.asm.org/.

These authors contributed equally.