title: 5'-UTR mediated translational control of splicing assembly factor RNP-4F expression during development of the Drosophila central nervous system.
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.
authors.affiliation: Department of Biology, Cell Molecular and Structural Biology Program, Miami University, Oxford, OH 45056, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
authors.forename: Julianne T
authors.initials: JT
authors.lastname: Yang
authors.forename: Dana L
authors.initials: DL
authors.lastname: Doctor
authors.forename: Bridgette A
authors.initials: BA
authors.lastname: Rawlins
authors.forename: B Colleen
authors.initials: BC
authors.lastname: Shields
authors.forename: Jack C
authors.initials: JC
authors.lastname: Vaughn
mesh_terms: D020121:5' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D002490:Central Nervous System / Q000254:growth & development*; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics* / Q000254:growth & development; D004625:Embryo, Nonmammalian / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D055029:Inverted Repeat Sequences; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D020121:5' Untranslated Regions; D029721:Drosophila Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; C100381:RNA-binding protein 4F, Drosophila; D012261:Ribonucleoproteins; D049452:Green Fluorescent Proteins
keywords: 3′-UTR; 3′-untranslated mRNA region; 5′-UTR; 5′-untranslated mRNA region; Alternative splicing; CNS; DNA complementary to RNA; Drosophila adenosine deaminase acting on RNA; GFP; GFP reporter; IRES; PCR; Posttranscriptional regulation; RNP-4F; RT-PCR; U-series small nuclear ribonucleoprotein; U-snRNP; base pair; bp; cDNA; central nervous system; dADAR; green fluorescent protein; internal ribosomal entry site; mRNA; messenger RNA; nt; nucleotide; polymerase chain reaction; reverse transcriptase-polymerase chain reaction; ribonucleoprotein encoded by rnp-4f gene in polytene band 4F; rnp-4f gene