EVIDENCE THAT U5 SNRNP RECOGNIZES THE 3'-SPLICE-SITE FOR CATALYTIC STEP-II IN MAMMALS

The first AG dinucleotide downstream from the branchpoint sequence (BP S) is chosen as the 3' splice site during catalytic step II of the spl icing reaction, The mechanism and factors involved in selection of thi s AG are not known. Early in mammalian spliceosome assembly, U2AF(65) binds to the pyrimidine tract between the BPS and AG. Here we show tha t U2AF(65) crosslinking is replaced by crosslinking of three proteins of 110, 116 and 220 kDa prior to catalytic step II, and we provide evi dence that all three proteins are components of U5 snRNP. These protei ns interact with pre-mRNA in the region spanning from immediately down stream of U2 snRNP's binding site at the BPS to just beyond the 3' spl ice site, We also demonstrate that there are strict constraints on bot h the sequence and the distance between the BPS and AG for catalytic s tep II, Together, these observations suggest that U5 snRNP is position ed on the 3' splice site by an interaction (direct or indirect) with U 2 snRNP bound at the BPS and by a direct interaction with the pyrimidi ne tract, The functional AG for catalytic step II may be specified, in turn, by its location with respect to the U5 snRNP binding site.