A tertiary interaction detected 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 base-paired structure within the spliceosome that is required for catalysis. Especially in light of significant similarities with group II self-splicing introns, we wished to investigate whether the purified RNAs might by themselves be able to form a complex similar to that which appears to exist in the spliceosome. To this end, we synthesized and purified large segments of human U2 and U6 snRNAs. Upon annealing, the two RNAs efficiently formed a stable and apparently extensively base-paired (Tm = 50-60 degrees C in the presence of 20 mM Mg2+) complex. To investigate possible tertiary interactions, we subjected the annealed complex to UV irradiation, and two crosslinked 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 Ia and opposite the invariant AGC in U6. Remarkably, this crosslink indicates a tertiary interaction essentially identical to one detected previously by genetic covariation in yeast. Together our results suggest that purified U2 and U6 snRNAs can anneal and fold to form a structure resembling that likely to exist in the catalytically active spliceosome.
Valadkhan
S
S
Department of Biological Science, Columbia University, New York, New York 10027, USA.
Manley
J L
JL
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D003432:Cross-Linking Reagents; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016914:Ribonuclease H; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D017381:Spliceosomes / Q000378:metabolism; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D012322:RNA Precursors; D012331:RNA, Fungal; D012342:RNA, Small Nuclear; D016914:Ribonuclease H