AN ELEMENT IN HUMAN U6 RNA DESTABILIZES THE U4 U6 SPLICEOSOMAL RNA COMPLEX/

Large-scale changes in RNA secondary structure, such as those that occ ur in some of the spliceosomal RNAs during pre-mRNA splicing, have bee n proposed to be catalyzed by ATP-dependent RNA helicases. Here we sho w that deproteinized human U4/U6 spliceosomal RNA complex, which has t he potential for extensive intermolecular base pairing, contains a cis -acting element that promotes its dissociation into free U4 and U6 RNA s. The destabilizing element corresponds to the base of a putative int ramolecular stem in U6 RNA that includes the 3' three-quarters of the molecule. Oligonucleotides expected to compete for U6 RNA 3' stem form ation promote assembly of the human U4/U6 RNA complex under conditions that otherwise result in dissociation of the U4/U6 complex. Truncatio n of the putative 3' stem-forming sequences in U6 RNA by oligonucleoti de-directed RNase H cleavage increases the melting temperature of the U4/U6 RNA complex by almost 20 degrees C, to a level commensurate with its intermolecular base-pairing potential. We conclude that the stabi lity of the competing human U6 RNA intramolecular 3' stem, combined wi th a low activation energy for conformational rearrangement, causes th e human U4/U6 RNA complex to be intrinsically unstable despite its bas e-pairing potential. Therefore, a helicase activity may not be necessa ry for disassembly of the human U4/U6 complex during activation of the spliceosome. We propose that a previously identified base-pairing int eraction between U6 and U2 RNAs may stabilize the human U4/U6 RNA comp lex by antagonizing U6 RNA 3' stem formation.