Poster abstracts

Poster number 27 submitted by Sushmita Ghosh

A novel role for full-length dADAR in 5'-UTR intron splicing regulation of rnp-4f pre-mRNA during Drosophila embryogenesis

G. Girija Lakshmi (Zoology, Cell and Molecular and Structural Biology Program, Miami University), Sushmita Ghosh (Zoology, Cell and Molecular and Structural Biology Program, Miami University), Gabriel Jones (Zoology, Cell and Molecular and Structural Biology Program, Miami University), Bridgette Rawlins (Zoology, Cell and Molecular and Structural Biology Program, Miami University), Jack Vaughn (Zoology, Cell and Molecular and Structural Biology Program, Miami University)

Abstract:
The Drosophila rnp-4f gene encodes a splicing assembly factor that dimerizes U4- and U6-snRNPs during spliceosome formation. 5’-UTR pre-mRNA intron processing results in two major isoforms, “long” (unspliced) and “short” (alternatively spliced). The long isoform has a secondary structure in which an intron pairs with adjacent highly evolutionarily-conserved exon 2 to form a stable 177-nt stem-loop. The coding potential for the two isoforms is identical, raising interesting questions as to the control mechanism and functional significance of this 5’-UTR intron splicing decision. It is known that the unspliced isoform localizes largely in the developing fly central nervous system, as do dADAR mRNAs, and that dADAR uses dsRNAs for substrate during deamination of particular adenosines to inosines in pre-mRNAs. These observations suggested an hypothesis in which dADAR protein may bind directly to the rnp-4f pre-mRNA stem-loop and inhibit splicing. To test this hypothesis, RNA electrophoretic mobility shift assay (REMSA) was carried out using in vitro transcribed stem-loop RNA incubated with embryo protein extract. Two RNA-protein complexes are detected by shifted RNA bands, suggesting involvement of two different proteins. Protein extract from a dADAR null mutant fly line results in only one shifted band, and recombinant dADAR results in a band shift. These observations suggest that dADAR protein may in part regulate intron splicing. To determine if unspliced mRNA levels are correlated with presence of dADAR protein during embryo development, qRT-PCR was carried out using protein extracts from wild-type and the dADAR mutant. A dramatic decrease in unspliced mRNA levels occurs in the dADAR mutant, a finding consistent with the REMSA results. These observations demonstrate a novel non-catalytic role for dADAR protein in rnp-4f 5’-UTR intron splicing regulation. We are now attempting to identify the regulatory protein(s) which bind to the stem-loop using MALDI-TOF technology.

Keywords: REMSA, alternative splicing regulation, non-catalytic dADAR function