Characterization of RNA binding specificity of the Drosophila sex-lethal protein by in vitro ligand selection.
The Drosophila sex-lethal (Sxl) protein, a regulator of somatic sexual differentiation, is an RNA binding protein with two potential RNA recognition motifs (RRMs). It is thought to exert its function on splicing by binding to specific RNA sequences within Sxl and transformer (tra) pre-mRNAs. To examine the Sxl RNA binding specificity in detail, we performed in vitro selection and amplification of ligand RNAs from a random sequence pool on the basis of affinity with Sxl protein. After three cycles of selection and amplification, we cloned and sequenced 17 cDNAs corresponding to the RNAs selected in vitro. Sequencing showed that most of the RNAs selected contain polyuridine stretches surrounded by purine residues. In vitro binding analysis revealed that the sequences of the in vitro selected RNAs with relatively high affinity for Sxl show similarity to that of the Sxl- and tra-regulated acceptor regions, including the invariant AG sequence for splicing. These results suggest that Sxl recognizes and preferentially binds to a polyuridine stretch with a downstream AG sequence.
Sakashita
E
E
Department of Biology, Faculty of Science, Kobe University, Japan.
Sakamoto
H
H
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000235:genetics*; D007301:Insect Hormones / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D011072:Poly U / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism*; D012326:RNA Splicing; D016601:RNA-Binding Proteins; D017423:Sequence Analysis, RNA
D018076:DNA, Complementary; D029721:Drosophila Proteins; D007301:Insect Hormones; D008024:Ligands; D016601:RNA-Binding Proteins; C067777:Sxl protein, Drosophila; D011072:Poly U; D012313:RNA