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Papers In Press, published online ahead of print July 20, 2006
Cellular Biochemistry, Max-Planck-Institut biophysikalische Chemie, Goettingen D-37077
Corresponding Author: Reinhard.Luehrmann{at}mpi-bpc.mpg.de
The kink-turn, a stem I–internal loop–stem II structure of the 5' stem-loop of U4 and U4atac snRNAs 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 analysed 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 pre bound 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.
J. Biol. Chem, 10.1074/jbc.M603350200
Submitted on April 7, 2006
Revised on July 14, 2006
Accepted on July 20, 2006
RNA structural requirements for the association of the spliceosomal hPrp31 protein with the U4 and U4atac snRNP
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