THE U5 AND U6 SMALL NUCLEAR RNAS AS ACTIVE-SITE COMPONENTS OF THE SPLICEOSOME

Five small nuclear RNAs (U1, U2, U4, U5, and U6) participate in precur sor messenger RNA (pre-mRNA) splicing. To probe their interactions wit hin the active center of the mammalian spliceosome, substrates contain ing a single photoactivatable 4-thiouridine residue adjacent to either splice site were synthesized, and crosslinks were induced during the course of in vitro splicing. An invariant loop sequence in U5 small nu clear RNA contacts exon 1 before and after the first step of splicing because a crosslink between U5 and the last residue of exon 1 appeared in the pre-mRNA and then in the cutoff exon 1 intermediate. Both of t hese crosslinked species could undergo subsequent splicing, indicating that the crosslinks reflect afunctional interaction that is maintaine d through both reaction steps. The same U5 loop aligns the two exons f or ligation since the first residue of exon 2 also became crosslinked to U5 in the lariat intermediate. An invariant sequence in U6 RNA beca me crosslinked to the conserved second position of the intron within b oth the lariat intermediate and the lariat intron product. On the basi s of these results, several conformational arrangements of small nucle ar RNAs within the spliceosomal active center can be distinguished, an d additional mechanistic parallels between the spliceosome and self-sp licing introns can be drawn.