The positional, structural, and sequence requirements of the Drosophila TLS RNA localization element.
The subcellular localization of mRNAs is a key step in the polarization of cells in organisms from yeast to man. Here, we use a transgenic fly/in situ hybridization assay system to define the positional, structural, and sequence requirements of the TLS, a stem loop RNA sequence element that mediates the subcellular localization of K10 and Orb transcripts in Drosophila oocytes. We find that the TLS is a highly robust and modular element. It mediates efficient RNA localization regardless of sequence context or position within the transcript. Site-specific mutagenesis experiments indicate that the size and shape of the stem and loop regions are critical determinants of TLS activity. Such experiments also identify specific base residues that are important for TLS activity. All such residues map to the stem portion of the structure. Significantly, mutations at these residues interfere with TLS activity only when they alter the stereochemistry of the stem's minor groove. For example, mutation of the A:U base pair at position 3 of the TLS stem to G:C severely reduces TLS activity, while mutation of the same base pair to U:A has no effect. Extensive searches for TLS-like elements in other Drosophila mRNAs using sequence and structural parameters defined by our experiments indicate that the TLS is unique to K10 and Orb mRNAs. This unexpected finding raises important questions as to how the many hundreds of other mRNAs that are known or thought to exhibit K10 and Orb-like localization are localized.
Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA. rcohen@ku.edu
Robert S
RS
Cohen
Sui
S
Zhang
Gretchen L
GL
Dollar
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D020029:Base Pairing; D001483:Base Sequence; D017124:Conserved Sequence; D004330:Drosophila / Q000737:chemistry* / Q000166:cytology / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004625:Embryo, Nonmammalian; D005260:Female; D017344:Genes, Insect; D017930:Genes, Reporter; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009710:Nucleotide Mapping; D009865:Oocytes / Q000378:metabolism; D010053:Ovary / Q000166:cytology; D017433:Protein Structure, Secondary; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors; D014158:Transcription, Genetic; D019076:Transgenes
D029721:Drosophila Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014157:Transcription Factors; C088225:fs(1)K10 protein, Drosophila; C089932:orb protein, Drosophila; D012313:RNA