Cross-linking of U2 snRNA using nitrogen mustard. Evidence for higher order structure.
Nuclear mRNA precursors are spliced by a large macromolecular complex called the spliceosome which contains, in most eucaryotes, five small nuclear RNAs (snRNAs) each in the form of a small ribonucleoprotein particle (the U1, U2, U5, and U4/U6 snRNPs). Although secondary structures have been derived for all five spliceosomal snRNAs based on phylogenetic, biochemical, and genetic data, little tertiary structure information is available. Here we use the general cross-linking reagent nitrogen mustard [bis-(2-chloroethyl)methylamine] to detect tertiary interactions within U2 snRNA. After the cross-linking of deproteinized HeLa nuclear extract, two intramolecularly cross-linked U2 species with anomalous electrophoretic mobility can be detected (X-U2#1 and X-U2#2). The 3' and 5' boundaries of each cross-link were determined by rapid enzymatic RNA sequencing of end-labeled RNA. X-U2#1 is cross-linked between the region U41-U55 and G105 or G106, X-U2#2 between U53 and G97 or G98. We then tested the ability of the two cross-linked species to bind snRNP proteins in vitro (in nuclear extract or S100) and in vivo (in Xenopus oocytes). X-U2#2 reconstituted efficiently both in vitro and in vivo but X-U2#1 did not, as judged by immunoprecipitation with antibodies specific for Sm- and U2-specific proteins. Since the cross-link in X-U2#2 involves the Sm binding site but does not block snRNP assembly, our data strongly suggest that the Sm binding site lies on the surface of the native snRNP.
Datta
B
B
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06510.
Weiner
A M
AM
D001483:Base Sequence; D015152:Blotting, Northern; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D008466:Mechlorethamine / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011233:Precipitin Tests; D012342:RNA, Small Nuclear / Q000378:metabolism*
D003432:Cross-Linking Reagents; D012342:RNA, Small Nuclear; D008466:Mechlorethamine