2 RNA-BINDING DOMAINS DETERMINE THE RNA-BINDING SPECIFICITY OF NUCLEOLIN

Nucleolin is an abundant nucleolar RNA-binding protein that seems to b e involved in many aspects of ribosome biogenesis. Nucleolin contains four copies of a consensus RNA-binding domain (CS-RBD) found in severa l other proteins. In vitro RNA-binding studies previously determined t hat nucleolin interacts specifically with a short RNA stem-loop struct ure. Taken individually, none of the four CS-RBDs interacts significan tly with the RNA target, but a peptide that contains the first two adj acent CS-RBDs (R12) is sufficient to account for nucleolin RNA-binding specificity and affinity. The full integrity of these two domains is required, since N- or C-terminal deletion abolishes the specific inter action with the RNA. Mutation of conserved amino acids within the RNP- 1 sequence of CS-RBD 1 or 2 drastically reduces the interaction with t he RNA, whereas mutation of the analogous residues in CS-RBDs 3 and 4 has no effect in the context of the R1234G protein (which corresponds to the C-terminal end of nucleolin). Our results demonstrate that nucl eolin RNA binding specificity is the result of a cooperation between t wo CS-RBDs (RBDs I and 2) and also suggests a direct or indirect invol vement of the RNP-1 consensus sequence of both CS-RBDs in the recognit ion of the RNA target.