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J. Biol. Chem., Vol. 281, Issue 38, 28278-28286, September 22, 2006

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RNA Structural Requirements for the Association of the Spliceosomal hPrp31 Protein with the U4 and U4atac Small Nuclear Ribonucleoproteins^*

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

The kink-turn, a stem I-internal loop-stem II structure of the 5 ' stem-loop of U4 and U4atac small nuclear (sn) RNAs bound by 15.5K protein is required for binding of human Prp31 protein (hPrp31) during U4 and U4atac snRNP assembly. In box C/D snoRNPs a similar kink-turn with bound 15.5K protein is required for selective binding of proteins NOP56 and NOP58. Here we analyzed RNA structural requirements for association of hPrp31 with U4 snRNP in vitro by hydroxyl radical footprinting. hPrp31 induced protection of the terminal penta-loop, as well as of stems I and II flanking the kink-turn. Similar protection was found with U4/U6 snRNA duplex prebound with 15.5K protein. A detailed mutational analysis of the U4 snRNA elements by electrophoretic mobility shift analysis revealed that stem I could not be shortened, although it tolerated sequence alterations. However, introduction of a third Watson-Crick base pair into stem II significantly reduced hPrp31 binding. While stem I of U4atac snRNA showed relaxed binding requirements, its stem II requirements were likewise restricted to two base pairs. In contrast, as shown previously, stem II of the kink-turn motif in box C/D snoRNAs is comprised of three base pairs, and NOP56 and NOP58 require a G-C pair at the central position. This indicates that hPrp31 binding specificity is achieved by the recognition of the two base pair long stem II of the U4 and U4atac snRNAs and suggests how discrimination is achieved by RNA structural elements during assembly of U4/U6 and U4atac/U6atac snRNPs and box C/D snoRNPs.

Received for publication, April 7, 2006 , and in revised form, July 14, 2006.

^* This work was supported by grants from the Deutsche Forschungsgemeinschaft (LU294/12-1), the Ernst-Jung-Stiftung, and the Fonds der Chemischen Industrie (to R. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2 and Refs. 1 and 2.

¹ These authors contributed equally to this work.

² Present address: University of Massachusetts Medical School, Program in Molecular Medicine, 373 Plantation St., Worcester, MA 01605.

³ To whom correspondence should be addressed: Dept. of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany. Tel.: 49-551-201-1405; Fax: 49-551-201-1197; E-mail: reinhard.luehrmann{at}mpi-bpc.mpg.de.

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