Cross-linking of U1 snRNA using nitrogen mustard. Evidence for higher order structure

Secondary structures for all five spliceosomal small nuclear (sn) RNAs (U1, U2, U4, U5, and U6 snRNAs) have been derived from phylogenetic, biochemical, and genetic data, but tertiary structure information has been more difficult to obtain. Here we have used the general cross-linking reagent nitrogen mustard (bis-(2-chloroethyl)methylamine) to explore the tertiary conformation of naked U1 snRNA. We detected two intramolecularly cross-linked U1 species (X-U1#1 and X-U1#2) after cross-linking of deproteinized HeLa nuclear extract. We determined the cross-linked sites and found that X-U1#1 is cross-linked between the C82-A85 and U129, while X-U1#2 is cross-linked between U105-G108 and A118. We then tested the ability of these two cross-linked species to bind small nuclear ribonucleo-protein (snRNP) proteins in vitro (in HeLa nuclear extract or S100) and in vivo (in Xenopus oocytes). Both X-U1#1 and X-U1#2 were found to reconstitute efficiently in vitro and in vivo, as judged by immunoprecipitation with antibodies specific for Sm and U1-specific proteins. Our data suggest that (i) the Sm-binding site lies on the surface of the native U1 snRNP, since the cross-link in X-U1#1 involves the Sm-binding site but does not block snRNP assembly, and (ii) U1 snRNA may adopt the correct tertiary conformation even in the absence of U1 snRNP proteins.