Poster abstracts
Poster number 67 submitted by Fatemeh Kohram
A heterodimer-based regulatory role for Drosophila truncated dADAR protein isoform function
Fatemeh Kohram (Biology Dept., Miami University), Sushmita Ghosh (Biology Dept., Miami University), Jack C. Vaughn (Biology Dept., Miami University)
Abstract:
The Drosophila rnp-4f gene encodes RNP-4F protein which is a splicing assembly factor. The Drosophila rnp-4f gene codes for two mRNA isoforms (“long” and “short”) which differ by a 177-nt sequence (caused by alternative splicing in the 5’-UTR within intron 0 and exon 2) that forms an evolutionarily conserved stem-loop. Several observations suggest that rnp-4f expression may be regulated by a feedback pathway. Northerns and RT-PCR studies suggest that there is a developmental switch that controls the levels of the two isoforms. Northerns show that the long mRNA isoform level peaks in the mid-embryo stage. Westerns and RT-PCR show that high levels of RNP-4F protein correspond to elevated levels of the short rnp-4f mRNA isoform during very early embryo stages and late stages of fly development. Evolutionary conservation of the rnp-4f stem-loop and developmental regulation of alternative transcript levels suggest functional significance of this 5’-UTR stem-loop structure. RNA electrophoretic mobility shift assay using in vitro transcribed RNA (rnp-4f 5’-UTR 177-nt stem-loop) and whole embryo protein extract from wild-type embryos and dADAR mutant embryo protein combined with qPCR analysis and RNAi studies suggest that dADAR is one of the two proteins that bind to the stem-loop. However, the identity of the other protein remains unknown. A structural study has revealed that there exists a conserved sequence on U6-snRNA to which the RNP-4F chaperone may bind. A stretch of 12 nucleotides within the 5’-UTR 177-nt in the rnp-4f mRNA shares significant sequence similarity with the conserved binding site on U6-snRNA. Another level of similarity is that in both cases the consensus sequence lies within a long stem-loop secondary structure. Here, we describe construction and utilization of several new rnp-4f gene expression study vectors using a GFP reporter in the Φ C31Based on all our findings we propose a model in which RNP-4F binds to the stem-loop in the rnp-4f mRNA 5’-UTR (long isoform) and regulates its own expression via a feedback pathway.
Keywords: dADAR, protein function