Dissection of the target specificity of the RNA-binding protein HOW reveals dpp mRNA as a novel HOW target.
Regulation of RNA metabolism plays a major role in controlling gene expression during developmental processes. The Drosophila RNA-binding protein Held out wing (HOW), regulates an array of developmental processes in embryonic and adult growth. We have characterized the primary sequence and secondary structural requirements for the HOW response element (HRE), and show that this site is necessary and sufficient for HOW binding. Based on this analysis, we have identified the Drosophila TGFbeta homolog, dpp, as a novel direct target for HOW negative regulation in the wing imaginal disc. The binding of the repressor isoform HOW(L) to the dpp 3' untranslated region (UTR) leads to a reduction of GFP-dpp3'UTR reporter levels in S-2 cells, in an HRE site-dependent manner. Moreover, co-expression of HOW(L) in the wing imaginal disc with a dpp-GFP fusion construct led to a reduction in DPP-GFP levels in a dpp-3'UTR-dependent manner. Conversely, a reduction of the endogenous levels of HOW by targeted expression of HOW-specific double-stranded RNA led to a corresponding elevation in dpp mRNA level in the wing imaginal disc. Thus, by characterizing the RNA sequences that bind HOW, we demonstrate a novel aspect of regulation, at the mRNA level, of Drosophila DPP.
Israeli
David
D
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
Nir
Ronit
R
Volk
Talila
T
D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004330:Drosophila / Q000196:embryology / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D049452:Green Fluorescent Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014921:Wings, Animal / Q000196:embryology / Q000378:metabolism*
D020413:3' Untranslated Regions; D017931:DNA Primers; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014157:Transcription Factors; C064196:dpp protein, Drosophila; C106296:how protein, Drosophila; C096359:sr protein, Drosophila; D049452:Green Fluorescent Proteins