An RNA electrophoretic mobility shift and mutational analysis of rnp-4f 5'-UTR intron splicing regulatory proteins in Drosophila reveals a novel new role for a dADAR protein isoform.

Alternative splicing greatly enhances the diversity of proteins encoded by eukaryotic genomes, and is also important in gene expression control. In contrast to the great depth of knowledge as to molecular mechanisms in the splicing pathway itself, relatively little is known about the regulatory events behind this process. The 5'-UTR and 3'-UTR in pre-mRNAs play a variety of roles in controlling eukaryotic gene expression, including translational modulation, and nearly 4000 of the roughly 14,000 protein coding genes in Drosophila contain introns of unknown functional significance in their 5'-UTR. Here we report the results of an RNA electrophoretic mobility shift analysis of Drosophila rnp-4f 5'-UTR intron 0 splicing regulatory proteins. The pre-mRNA potential regulatory element consists of an evolutionarily-conserved 177-nt stem-loop arising from pairing of intron 0 with part of adjacent exon 2. Incubation of in vitro transcribed probe with embryo protein extract is shown to result in two shifted RNA-protein bands, and protein extract from a dADAR null mutant fly line results in only one shifted band. A mutated stem-loop in which the conserved exon 2 primary sequence is changed but secondary structure maintained by introducing compensatory base changes results in diminished band shifts. To test the hypothesis that dADAR plays a role in intron splicing regulation in vivo, levels of unspliced rnp-4f mRNA in dADAR mutant were compared to wild-type via real-time qRT-PCR. The results show that during embryogenesis unspliced rnp-4f mRNA levels fall by up to 85% in the mutant, in support of the hypothesis. Taken together, these results demonstrate a novel role for dADAR protein in rnp-4f 5'-UTR alternative intron splicing regulation which is consistent with a previously proposed model.

Spatial and temporal expression of dADAR mRNA and protein isoforms during embryogenesis in Drosophila melanogaster.

Adenosine Deaminases Acting on RNA (ADARs) function to co-transcriptionally deaminate specific (or non-specific) adenosines to inosines within pre-mRNAs, using double-stranded RNAs as substrate. In both Drosophila and mammals, the best-studied ADAR functions are to catalyze specific nucleotide conversions within mRNAs encoding various ligand- or voltage-gated ion channel proteins within the adult brain. In contrast, ADARs within developing fly embryos have scarcely been studied, in part because they contain little or no editase activity, raising interesting questions as to their functional significance. Quantitative RT-PCR shows that two major developmentally regulated mRNA isoform classes are produced (full-length and truncated), which arise by alternative splicing and also alternative 3'-end formation. In situ localization of specific dADAR mRNA isoforms during embryogenesis reveals that the full-length class is found primarily within the developing germ band and central nervous system, whereas the truncated isoform is mostly located in gut endothelium. Developmental Western immunoblots show that both isoform classes are expressed into protein during embryogenesis. Both the rnp-4f 5'-UTR unspliced isoform and the full-length dADAR mRNA primarily localize in the embryonic germ band and subsequently throughout the developing central nervous system. Previous studies have shown that some rnp-4f pre-mRNAs are extensively edited by dADAR in the adult brain. Computer predictions suggest that intron-exon pairing promotes formation of an evolutionarily conserved secondary structure in the rnp-4f 5'-UTR, forming a 177-nt RNA duplex resembling an editing site complementary sequence, which is shown to be associated with splicing failure and to generate a long isoform. Taken together, these observations led us to explore the possibility that interaction between rnp-4f pre-mRNA and nuclear full-length dADAR protein may occur during embryogenesis. In dADAR null mutants, rnp-4f 5'-UTR alternative splicing is significantly diminished, suggesting a non-catalytic role for dADAR in splicing regulation. A working model is proposed which provides a possible molecular mechanism.