Establishment of an in vitro trans-splicing system in Trypanosoma brucei that requires endogenous spliced leader RNA.

ABSTRACT: In trypanosomes a 39 nucleotide exon, the spliced leader (SL) is donated to all mRNAs from a small RNA, the SL RNA, by trans-splicing. Since the discovery of trans-splicing in trypanosomes two decades ago, numerous attempts failed to reconstitute the reaction in vitro. In this study, a crude whole-cell extract utilizing the endogenous SL RNA and synthetic tubulin pre-mRNA were used to reconstitute the trans-splicing reaction. An RNase protection assay was used to detect the trans-spliced product. The reaction was optimized and shown to depend on ATP and intact U2 and U6 snRNPs. Mutations introduced at the polypyrimidine tract and the AG splice site reduced the reaction efficiency. To simplify the assay, RT-PCR and quantitative real-time PCR assays were established. The system was used to e

SUBMITTER: Shaked H 

PROVIDER: S-EPMC2879531 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature