Poster abstracts
Poster number 39 submitted by Sushmita Ghosh
Regulation of expression of RNP-4F splicing assembly factor in Drosophila melanogaster
Sushmita Ghosh (Dept. of Biology, Miami University), Shelby E. Thomas (Dept. of Biology, Miami University), Lindsey M. Abraham (Dept. of Biology, Miami University), Jack C. Vaughn (Dept. of Biology, Miami University)
Abstract:
The Drosophila rnp-4f gene encodes RNP-4F protein which is a splicing assembly factor. 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 ɸC31 system. The results confirm our previous observation that presence of the regulatory stem-loop enhances RNP-4F protein expression. However, in that study, the enhancement factor protein was not identified. We show here that overexpression of RNP-4F transgene results in additional translation, as indicated by the GFP reporter in the fluorescent images. Based 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: rnp-4f gene, UAS-GAL4 system, expression control