A tertiary interaction detested in a human U2-U6 snRNA complex assembled in vitro resembles a genetically proven interaction in yeast

U2 and U6 small nuclear RNAs are thought to play critical roles in pre-mRNA splicing catalysis, Genetic evidence suggests they form an extensively bas e-paired structure within the spliceosome that is required for catalysis. E specially in light of significant similarities with group II self-splicing introns, we wished to investigate whether the purified RNAs might by themse lves be able to form a complex similar to that which appears to exist in th e spliceosome. To this end, we synthesized and purified large segments of h uman U2 and U6 snRNAs. Upon annealing, the two RNAs efficiently formed a st able and apparently extensively base-paired (T-m = 50-60 degrees C in the p resence of 20 mM Mg2+) complex. To investigate possible tertiary interactio ns, we subjected the annealed complex to UV irradiation, and two crosslinke d species were identified and characterized, The major one links the second G in the highly conserved and critical ACAGAGA sequence in U6 with an A in U2 just 5' to U2-U6 helix la and opposite the invariant AGC in U6. Remarka bly, this crosslink indicates a tertiary interaction essentially identical to one detected previously by genetic covariation in yeast, Together our re sults suggest that purified U2 and U6 snRNAs can anneal and fold to form a structure resembling that likely to exist in the catalytically active splic eosome.