A specific RNA hairpin loop structure binds the RNA recognition motifs of the Drosophila SR protein B52.
B52, also known as SRp55, is a member of the Drosophila melanogaster SR protein family, a group of nuclear proteins that are both essential splicing factors and specific splicing regulators. Like most SR proteins, B52 contains two RNA recognition motifs in the N terminus and a C-terminal domain rich in serine-arginine dipeptide repeats. Since B52 is an essential protein and is expected to play a role in splicing a subset of Drosophila pre-mRNAs, its function is likely to be mediated by specific interactions with RNA. To investigate the RNA-binding specificity of B52, we isolated B52-binding RNAs by selection and amplification from a pool of random RNA sequences by using full-length B52 protein as the target. These RNAs contained a conserved consensus motif that constitutes the core of a secondary structural element predicted by energy minimization. Deletion and substitution mutations defined the B52-binding site on these RNAs as a hairpin loop structure covering about 20 nucleotides, which was confirmed by structure-specific enzymatic probing. Finally, we demonstrated that both RNA recognition motifs of B52 are required for RNA binding, while the RS domain is not involved in this interaction.
Shi
H
H
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA.
Hoffman
B E
BE
Lis
J T
JT
D000818:Animals; D001483:Base Sequence; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000235:genetics*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D010750:Phosphoproteins / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins / Q000378:metabolism*; D013329:Structure-Activity Relationship
D029721:Drosophila Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; C070791:B52 protein, Drosophila; D012313:RNA