THE CANONICAL GU DINUCLEOTIDE AT THE 5'-SPLICE-SITE IS RECOGNIZED BY P220 OF THE U5 SNRNP WITHIN THE SPLICEOSOME

Specific recognition of the 5' splice site (5'SS) by the spliceosome c omponents was studied using a simple in vitro system in which a short 5'SS RNA oligonucleotide specifically induces the assembly of snRNP pa rticles into spliceosome-like complexes and actively participates in a trans-splicing reaction. Short-range crosslinking demonstrates that a U5 snRNP protein component, p220 (the human analogue of the yeast Prp 8) specifically interacts with the invariant GU dinucleotide at the 5' end of the intron. The GU:p220 interaction can be detected in the fun ctional splicing complex B. Although p220 has been known to contact se veral nucleotides around the 5' splice junction, the p220:GU dinucleot ide interaction described here is remarkably specific. Consistent with the high conservation of the GU, even minor modifications of this ele ment affect recognition of the 5'SS RNA by p220. Substitution of uridi ne at the GU with base analogues containing a large methyl or iodo gro up, but not a smaller fluoro group at base position 5, interferes with association of 5'SS RNA with snRNP complexes and their functional par ticipation in splicing.