PROBING OF THE SPLICEOSOME WITH SITE-SPECIFICALLY DERIVATIZED 5' SPLICE-SITE RNA OLIGONUCLEOTIDES

We have developed a site-specific chemical modification technique to i ncorporate a photoreactive azidophenacyl (APA) group at designated int ernal positions along the RNA phosphodiester backbone. Using this tech nique, we have analyzed interactions of the 5' splice site (5'SS) RNA within the spliceosome. Several crosslinked products can be detected w ithin complex B using the derivatized 5'SS RNAs, including U6 snRNA, h Prp8p, and 114-, 90-, 70-, 54-, and 27-kDa proteins. The 5'SS RNAs der ivatized at intron positions +4 to +8 crosslink to Us snRNA, confirmin g the previously reported pairing interaction between these sequences. hPrp8p and p70 are crosslinked to the 5'SS RNA when the APA is placed within the 5' exon. Finally, a set of unidentified proteins, includin g p114, p54, and p27, is detected with the 5'SS RNA derivatized at int ron positions +4 to +8. Introduction of the bulky APA group near the 5 'SS junction (positions -2 to +3) strongly interferes with complex B f ormation and thus no APA crosslinks are observed at these positions. T ogether with our earlier observation that hPrp8p crosslinks to the GU dinucleotide at the 5' end of the intron, these results suggest that t he inhibitory effect of APA results from steric hindrance of the hPrp8 p:5'SS interaction. Unexpectedly, thio-modifications within the region of the 5'SS RNA that is involved in base pairing to U6 snRNA strongly stimulate complex B formation.