Isolation and partial characterization of U1-U6 small RNAs from Bombyx mori

Abstract

We have used a variety of techniques to characterize the U-series small nuclear RNAs from the posterior silk gland of Bombyx mori. Six molecular species have been identified which correspond to the vertebrate U1-U6 RNAs by the following criteria: (a) presence of the RNAs in ribonucleoprotein particles which can be immunoprecipitated by lupus Sm antisera; (b) presence of a 2,2,7-trimethylguanosine cap, as assayed by immunoprecipitation with anti-2,2,7-trimethylguanosine IgG; (c) size, as assayed by acrylamide/urea gel electrophoresis using HeLa cell U-RNA markers; and (d) primary nucleotide sequence, as determined by chemical/enzymatic cleavage of end-labeled molecules. The high conservation of primary sequence (66-81% homology based on partial sequences) relative to the corresponding vertebrate U-RNAs has permitted unambiguous identification of each molecule. With the exception of two subspecies of U3 RNA, the U-snRNAs of Bombyx exhibit a striking conservation of secondary structure relative to the proposed structures of the U-RNAs of vertebrates. This conservation is best exemplified by several compensatory base alterations that result in the maintenance of hairpin structures. These are particularly evident in U1 and U5 RNAs. Bombyx U3 is interesting in that two subspecies (of a total of four that were sequenced) diverge considerably in sequence (and presumably in structure) relative to the U3 RNA of vertebrates. The most abundant U-RNAs in the posterior silk gland appear to be U1 and U2, while U3-U6 are present in relatively small amounts.