Title

Genetic interactions between splicing and RNA editing in Drosophila

Date of Completion

January 2004

Keywords

Biology, Molecular|Biology, Genetics

Degree

Ph.D.

Abstract

Previous work has shown that one region of the para transcription unit requires MLE for normal processing of the transcript (Reenan et al, 2000). This particular region of para contains three A-to-I editing sites and a predicted ECS that forms a duplex with the editing site sequence to target the editing enzyme, dADAR, to the substrate. The mutant helicase, MLEnapts, causes skipping of the edited exon of para. We hypothesized that since RNA editing requires a RNA secondary structure, the MLE helicase is necessary to facilitate resolution of the structure at this region of para. ^ To study the interactions of RNA editing and splicing at this site in para, experiments were designed to (1) identify the mutation(s) in mle that cause the napts phenotype and (2) identify cis-acting suppressors of the mle napts phenotype. ^ Two potential mutations were identified, a 7 bp insertion in the intron and T415S mutation near the ATPase motif. Wild type and mle napts transgenes were made. Wild type transgenic flies were able to complement male lethality associated with the null allele of mle and reduce the skipping of the edited exon of para in a mlenapts background. The mle napts transgene did not complement male lethality associated with the null allele of mle or cause skipping of the edited exon indicating that the MLEnapts protein was not being expressed. We, therefore, were unable to confirm the mlenapts mutation. ^ A mini reporter construct was created to recapitulate the skipping that occurs in a mlenapts background in order to find cis-acting suppressors. Within this reporter construct three conserved elements were found using comparative sequence analysis of other Drosophilidae species: the ECS, DCS (donor site complementary sequence), and a hairpin (HP). These elements were deleted individually or in combination to determine the effect of MLEnapts. Deletion of the HP significantly reduced the skipping while deletions of the ECS and DCS did not. Reporter constructs that deleted all three conserved elements and deletion of the DCS and HP together were the only complete suppressors of the skipping, indicating that the HP is responsible for the skipping. ^