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

Secondary structures for all five spliceosomal small nuclear (sn) RNAs (Ul, U2, U4, US, 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-chloroethy1)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-Ul#1 is cross-linked between the Cs2-As6 and U"', while X-U1#2 is cross-linked between U1Os-G'OS and A"'.We then tested the ability of these two cross-linked species to bind small nuclear ribonucleoprotein (snRNP) proteins in vitro (in HeLa nuclear extract or 5100) and in vivo (inxenopus oocytes).Both X-Ul#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 Ul-spe- cific 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-Ul#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.