Two different combinations of RNA-binding domains determine the RNA binding specificity of nucleolin

Nucleolin is an abundant nucleolar protein involved in several steps of rib osome biogenesis. The protein is highly conserved through evolution and pos sesses four RNA-binding domains (RBD), which are likely to determine its RN A binding specificity. Previous studies have shown that nucleolin interacts with two different RNA targets. The first is a small stem-loop structure, the nucleolin recognition element (NRE), found all along the pre-ribosomal RNA. The second is a short single-stranded RNA sequence, the evolutionary c onserved motif (ECM), located five nucleotides downstream of the first proc essing site in the pre-ribosomal RNA 5' external transcribed spacer. Bioche mical, genetic, and structural studies have shown that the first two RBD of nucleolin are necessary and sufficient for the specific interaction of nuc leolin with the NRE motif. In this work, we have studied the interaction of nucleolin with the ECM sequence. Deletion and mutational analyses showed t hat all four RBDs of hamster nucleolin were required for the interaction wi th the ECM sequence. This RNA binding specificity is conserved between hams ter and Xenopus laevis, whereas the Xenopus protein does not interact with the NRE. Nucleolin is the first example of a protein that requires four RBD s for its interaction with an RNA target, demonstrating that a single prote in can use different combinations of RED to interact specifically with seve ral RNA sequences.