Natural Antisense and RNP-4F Expression in Drosophila

Miami University Oxford, Oxford OH

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Abstract

DESCRIPTION (provided by applicant): Drosophila RNP4F is an ortholog to human p110, which functions with the pre-mRNA processing (PRP) protein class as a recycling factor to carry U4 and U6-snRNPs to the assembling spliceosome. Both inherited and acquired defects in premRNA processing are being increasingly recognized as causes of human diseases. A notable example of a relatively common (and also relevant to this proposal) human genetic disease which has been shown to be due to mutations in three different PRP genes is retinitis pigmentosa (RP), affecting 1 in 4000 individuals, being characterized by progressive retinal degeneration and eventually total blindness. Our previous work has shown that rnp-4f mRNAs undergo alternative splicing; which suggests that RNP-4F may have other functions, perhaps related to those carded out by the fully spliced gene product. We outline plans to characterize alternative RNP-4F proteins and their functions, and anticipate that the results may lead to discovery of corresponding alternative p110 functions in humans. Specifically, we propose to: (a) fully identify all rnp.4f transcript isoforms and determine their in situ localizations; (b) determine in situ localizations for the correspondingly encoded proteins; (c) eliminate each mRNA isoform specifically using RNAi in an attempt to determine functions for the encoded RNP-4F isoforms; and (d) identify neighbor proteins for the different RNP-4F isoforms via yeast two-hybrid screens. We have previously reported existence of extensively A-to-G edited rnp-4f mRNA in an adult head cDNA clone, which was subsequently shown to have arisen due to the action of dADAR editase on double-stranded RNA in which the antisense strand was supplied by a developmentally regulated readthrough transcript class of the sas10 gene. A decline in rnp-4f mRNA levels was observed to accompany rising readthrough transcript levels, suggesting a role for the antisense RNA in posttranscriptional regulation of rnp-4f gene expression during development. In this proposal, we outline plans to determine the relationship between long natural antisense transcripts and gene expression control in the Drosophila rnp-4f/sas10 model gene pair. We will do this by: (a) showing that sas10 readthrough transcripts are required for the observed decline in rnp-4f mRNA levels; (b) showing that sas10 readthrough transcripts co-localize with rnp-4f mRNAs; and (c) evaluating mechanisms for rnp-4f decay: (1) dADAR editase transcript modification followed by degradation and (2) destruction via the RNAi pathway. This research will provide a basis for understanding the role of long natural antisense RNAs in posttranscriptional control of eukaryotic gene expression, and may provide a model for elucidation of roles for comparable RNAs in organisms including humans, estimated to contain about 800 such transcripts. This work will also lead: to elucidation of alternative RNP-4F functions: perhaps to discovery of corresponding alternative p110 functions in humans.