Binding of the 60-kDa Ro autoantigen to Y RNAs: Evidence for recognition in the major groove of a conserved helix
CYNTHIA D. GREEN a1 p1 , KATHERINE S. LONG a1 , HONG SHI a1 and SANDRA L. WOLIN a1 c1
a1 Departments of Cell Biology and Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510, USA
The 60-kDa Ro autoantigen is normally complexed with small cytoplasmic RNAs known as Y RNAs. In Xenopus oocytes, the Ro protein is also complexed with a large class of variant 5S rRNA precursors that are folded incorrectly. Using purified baculovirus-expressed protein, we show that the 60-kDa Ro protein binds directly to both Y RNAs and misfolded 5S rRNA precursors. To understand how the protein recognizes these two distinct classes of RNAs, we investigated the features of Y RNA sequence and structure that are necessary for protein recognition. We identified a truncated Y RNA that is stably bound by the 60-kDa Ro protein. Within this 39-nt RNA is a conserved helix that is proposed to be the binding site for the Ro protein. Mutagenesis of this minimal Y RNA revealed that binding by the 60-kDa Ro protein requires specific base pairs within the conserved helix, a singly bulged nucleotide that disrupts the helix, and a three-nucleotide bulge on the opposing strand. Chemical probing experiments using diethyl pyrocarbonate demonstrated that, in the presence of the two bulges, the major groove of the conserved helix is accessible to protein side chains. These data are consistent with a model in which the Ro protein recognizes specific base pairs in the conserved helix by binding in the major groove of the RNA. Furthermore, experiments in which dimethyl sulfate was used to probe a naked and protein-bound Y RNA revealed that a structural alteration occurs in the RNA upon Ro protein binding.(Received October 20 1997)
(Revised November 20 1997)
(Accepted April 6 1998)
Key Words: RNA–protein interactions; RNA structure; Ro RNPs; small RNAs.
c1 Reprint requests to: Sandra L. Wolin, Departments of Cell Biology and Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, 295 Congress Avenue, New Haven, Connecticut 06510, USA; e-mail: firstname.lastname@example.org.
p1 Present address: CuraGen Corporation, 555 Long Wharf Drive, New Haven, Connecticut 06511, USA.