Functional interaction of a novel 15.5kD [U4/U6.U5] tri-snRNP protein with the 5' stem-loop of U4 snRNA.

Activation of the spliceosome for splicing catalysis requires the dissociation of U4 snRNA from the U4/U6 snRNA duplex prior to the first step of splicing. We characterize an evolutionarily conserved 15.5 kDa protein of the HeLa [U4/U6·U5] tri‐snRNP that binds directly to the 5′ stem–loop of U4 snRNA. This protein shares a novel RNA recognition motif with several RNP‐associated proteins, which is essential, but not sufficient for RNA binding. The 15.5kD protein binding site on the U4 snRNA consists of an internal purine‐rich loop flanked by the stem of the 5′ stem–loop and a stem comprising two base pairs. Addition of an RNA oligonucleotide comprising the 5′ stem–loop of U4 snRNA (U4SL) to an in vitro splicing reaction blocked the first step of pre‐mRNA splicing. Interestingly, spliceosomal C complex formation was inhibited while B complexes accumulated. This indicates that the 15.5kD protein, and/or additional U4 snRNP proteins associated with it, play an important role in the late stage of spliceosome assembly, prior to step I of splicing catalysis. Our finding that the 15.5kD protein also efficiently binds to the 5′ stem–loop of U4atac snRNA indicates that it may be shared by the [U4atac/U6atac·U5] tri‐snRNP of the minor U12‐type spliceosome.