Characterization of the catalytic activity of U2 and U6 snRNAs.
Removal of introns from pre-messenger RNAs in eukaryotes is carried out by the spliceosome, an assembly of a large number of proteins and five small nuclear RNAs (snRNAs). We showed previously that an in vitro transcribed and assembled base-paired complex of U2 and U6 snRNA segments catalyzes a reaction that resembles the first step of splicing. Upon incubation with a short RNA oligonucleotide containing the consensus sequence of the pre-mRNA branch site, the U2/U6 complex catalyzed a reaction between the 2' OH of a bulged adenosine and a phosphate in the catalytically important AGC triad of U6, leading to the formation of an X-shaped product, RNA X, apparently linked by an unusual phosphotriester bond. Here we characterize this splicing-related reaction further, showing that RNA X formation is an equilibrium reaction, and that the low yield of the reaction likely reflects an unfavorable equilibrium coefficient. Consistent with a phosphotriester linkage, RNA X is highly alkali-sensitive, but only mildly acid-sensitive. We also show that mutations in the AGC sequence of U6 can have significant effects on RNA X formation, further extending the similarities between splicing and RNA X formation. We also demonstrate that pseudouridylation of U2 enhances RNA X formation, and that U6 snRNA purified from nuclear extracts is capable of forming RNA X. Our data suggest that the ability to form RNA X might be an intrinsic property of spliceosomal snRNAs.
Valadkhan
Saba
S
Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
Manley
James L
JL
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D012342:RNA, Small Nuclear; C405484:U2 small nuclear RNA; C405486:U6 small nuclear RNA