1. 5'-UTR mediated translational control of splicing assembly factor RNP-4F expression during development of the Drosophila central nervous system.
- Author
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Chen J, Yang JT, Doctor DL, Rawlins BA, Shields BC, and Vaughn JC
- Subjects
- 5' Untranslated Regions, Animals, Base Sequence, Drosophila Proteins metabolism, Drosophila melanogaster growth & development, Embryo, Nonmammalian metabolism, Genes, Reporter, Green Fluorescent Proteins biosynthesis, Green Fluorescent Proteins genetics, Inverted Repeat Sequences, Microscopy, Fluorescence, Molecular Sequence Data, Protein Isoforms genetics, Protein Isoforms metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, Ribonucleoproteins metabolism, Central Nervous System growth & development, Drosophila Proteins genetics, Drosophila melanogaster genetics, Gene Expression Regulation, Developmental, Protein Biosynthesis, Ribonucleoproteins genetics
- Abstract
Drosophila RNP-4F is a highly conserved protein from yeast to human and functions as a spliceosome assembly factor during pre-mRNA splicing. Two major developmentally regulated rnp-4f mRNA isoforms have been described during fly development, designated "long" and "short," differing by a 177-nt tract in the 5'-UTR. This region potentially folds into a single long stable stem-loop by pairing of intron 0 and part of exon 2. Since the coding potential for the two isoforms is identical, the interesting question arises as to the functional significance of this evolutionarily-conserved 5'-UTR feature. Here we describe the effects of wild-type and mutated stem-loop on modulation of rnp-4f gene expression in embryos using a GFP reporter assay. In this work, a new GFP expression vector designated pUAS-Neostinger was constructed. The UAS-GAL4 system was utilized to trigger GFP expression using tissue-specific promoter driver fly lines. Fluorescence microscopy visualization, Western blotting and real-time qRT-PÇR were used to study and quantify GFP reporter protein and mRNA levels. A significant increase in GFP reporter protein expression due to presence of the wild-type stem-loop sequence/structure was unexpectedly observed with no concomitant increase in GFP reporter mRNA levels, showing that the 177-nt region enhancement acts posttranscriptionally. The effects of potential cis-acting elements within the stem-loop were evaluated using the reporter assay in two mutant constructs. Results of GFP reporter over-expression show that RNP-4F translational regulation is highly sensitive in the developing fly central nervous system. The potential molecular mechanism behind the observed translational enhancement is discussed., (© 2013.)
- Published
- 2013
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