NMR analysis of the hydrogen bonding interactions of the RNA-binding domains of the Drosophila sex-lethal protein with target RNA fragments with site-specific [3-15N]uridine substitutions.
It has been reported that a 183 residue fragment, consisting of the two RNA-binding domains (RBD1- RBD2) of the Drosophila melanogster Sex-lethal (Sxl) protein, strongly binds an oligonucleotide of the target RNA sequence (5'-GUUUUUUUUC-3') that regulates alternative splicing, and forms four or five hydrogen bonds with the imino groups of the RNA. In the present study, we used site-directed mutagenesis to improve the solubility of the didomain fragment of Sxl, and confirmed that this mutant fragment forms hydrogen bonds with the target RNA in the same manner as that of the wild-type fragment. The mutant fragment was shown to bind the cognate RNA sequences GUUUUUUUUC and AUUUUUUUUC more tightly than UUUUUUUUC. By using a [3-15N]uridine phosphoramidite, we synthesized a series of15N-labeled target RNAs, in which one of the uridine residues was specifically replaced by [3-15N]uridine. By observing the imino1H-15N coupling of the labeled uridine residue, we assigned all four of the hydrogen-bonded imino protons to U1, U2, U5 and U6, respectively, of the target RNA. The imino protons of U2 and U6 exhibited nuclear Overhauser effects with aliphatic protons of the protein. All these results indicate that the A/G, U1, U2, U5 and U6 residues in the target sequence of (G/A)UUUUUUUU are specifically recognized by the two RNA-binding domains of the Sxl protein.
Kim
I
I
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan.
Muto
Y
Y
Inoue
M
M
Watanabe
S
S
Kitamura
A
A
Yokoyama
S
S
Hosono
K
K
Takaku
H
H
Ono
A
A
Kainosho
M
M
Sakamoto
H
H
Shimura
Y
Y
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D006860:Hydrogen Bonding; D007301:Insect Hormones / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes; D009843:Oligoribonucleotides / Q000737:chemistry* / Q000378:metabolism; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D014529:Uridine
D029721:Drosophila Proteins; D007301:Insect Hormones; D009587:Nitrogen Isotopes; D009843:Oligoribonucleotides; D016601:RNA-Binding Proteins; C067777:Sxl protein, Drosophila; D012313:RNA; D014529:Uridine