SITE-SPECIFIC CROSS-LINKS OF YEAST U6 SNRNA TO THE PRE-MESSENGER-RNA NEAR THE 5'-SPLICE-SITE

We have introduced a single photochemical crosslinking reagent into sp ecific sites in the central domain of U6 to identify the sites that ar e in close proximity to the pre-mRNA substrate, Four distinct U6 snRNA s were synthesized with a single 4-thiouridine (4-thioU) at positions 46, 51, 54, and 57, respectively. Synthetic U6 RNA containing the 4-th ioU modifications can functionally reconstitute splicing activity in c ell-free yeast splicing extracts depleted of endogenous U6 snRNA, Upon photoactivation with UV (>300 nm), 4-thioU at position 46 forms cross links to pre-mRNA near the 5' splice site at nt +4, +5, +6, and +7 in the intron, whereas 4-thioU at position 51 crosslinks to the pre-mRNA at positions -2, -1, +1, +2, +3, and at the invariant G in the lariat intermediate. All crosslinks are dependent on the presence of ATP and the splicing substrate, The two crosslinks to the pre-mRNA from positi on 46 and 51 of U6 can also occur in prp2 heat-inactivated yeast splic ing extracts blocked immediately prior to the first chemical step. Sig nificantly, the crosslink from position 51 can undergo subsequent spli cing when the mutant extract is complemented with functional Prp2 prot ein in a chase experiment, indicating that the crosslink reflects a fu nctional interaction that is maintained during the first step. The cro sslink to lariat intermediate appears when the mutant spliceosomes are complemented with functional Prp2 protein added exogenously. This exp eriment is a paradigm for future studies in which different mutant ext racts are used to establish the stage in assembly at which particular RNA-RNA interactions defined by unique crosslinks occur.