A library of trimethylguanosine-capped small RNAS in Physarum polycephalum

Abstract

We have constructed a cDNA library for the trimethylguanosine-capped small RNAs (sRNAs) in the acellular slime mold Physarum polycephalum. Capped sRNAs were purified from total cellular RNA of vegetative microplasmodia by preparative immunoprecipitation with anti-trimethylguanosine antibody. The purified RNA was analyzed by polyacrylamide gel electrophoresis. Approx. eleven different capped sRNAs were observed with a size range of 70–204 nucleotides (nt). Based on their approximate sizes, the presence of a trimethylguanosine cap, and the presence of a lupus type-Sm antigen, molecules U1-U7 (excluding U3) were identified. Further confirmation of the identity of molecule U la was established by Northern hybridization, U4a by colony hybridization, and U6 and U7a by direct chemical sequence analysis. Purified capped sRNAs were tailed with oligo(A), and inserted into oligo(dT)-tailed plasmid pCDVl. The cDNAs were used to transform Escherichia coli strain HB101. Approx. 1.9 × 10 5 ampicillin-resistant (Ap R) transformants were obtained per μg of tailed sRNA. Dot-blot hybridization, using Physarum RNA precipitated with anti-cap antibody as a probe, indicated that approx. 94 % of the Ap R colonies contained recombinant DNAs. The library was screened by colony hybridization using heterologous sRNA probes. Clones hybridizing with heterologous sRNAs U1, U2, U4 and U7 were each represented in the library in approximately the same frequency as their relative abundance in the Physarum sRNA population they were derived from. The insert of one Physarum U4 clone was sequenced and was found to have 57.1% homology with nt 1–91 of the published sequence for rat U4 RNA. A 12-nt ‘functional’ subdomain of the rat U4 molecule was 83.3% conserved in Physarum U4.