Two Different Combinations of RNA-binding Domains Determine the RNA Binding Specificity of Nucleolin

Hervé Ginisty,François Amalric,Philippe Bouvet

doi:10.1074/jbc.m011120200

Abstract

Nucleolin is an abundant nucleolar protein involved in several steps of ribosome biogenesis. The protein is highly conserved through evolution and possesses four RNA-binding domains (RBD), which are likely to determine its RNA 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 conserved motif (ECM), located five nucleotides downstream of the first processing site in the pre-ribosomal RNA 5' external transcribed spacer. Biochemical, genetic, and structural studies have shown that the first two RBD of nucleolin are necessary and sufficient for the specific interaction of nucleolin with the NRE motif. In this work, we have studied the interaction of nucleolin with the ECM sequence. Deletion and mutational analyses showed that all four RBDs of hamster nucleolin were required for the interaction with the ECM sequence. This RNA binding specificity is conserved between hamster and Xenopus laevis, whereas the Xenopus protein does not interact with the NRE. Nucleolin is the first example of a protein that requires four RBDs for its interaction with an RNA target, demonstrating that a single protein can use different combinations of RBD to interact specifically with several RNA sequences.

Highlights

Specific RNA-protein interactions play an important role in gene expression
To determine whether the same RNA-binding domains were involved in the specific recognition of the evolutionary conserved motif (ECM) motif, we compared the interaction of the nucleolin recognition element (NRE) and the ECM sequences with several recombinant proteins, which contain different numbers of nucleolin RBD (Fig. 1A)
No cross-linking was observed between RNA645/677 and R12, whereas the R1234 protein interacts with this RNA. This experiment suggests that different combinations of RBDs are used for the specific interaction with the NRE and the ECM motifs

Summary

Introduction

Specific RNA-protein interactions play an important role in gene expression. One of the most common protein sequence motifs involved in these interactions is the RNA-binding domain (RBD), called the RNA recognition motif (RRM) [1, 2]. These are the single RBDs from the U1A and U2BЈЈ protein bound to a stem-loop structure [8, 17, 18], the two RBDs of Sex-lethal, hnRNPA1, and poly(A)-binding protein bound to a singlestranded RNA [12, 16, 19], and the first two RBD of nucleolin bound to a stem-loop structure [20] These different RNAprotein complexes revealed that the RBD uses the highly conserved RNP1 and RNP2 motifs in addition to the more highly divergent loops and linker regions specific to each RBD for the specific binding with their respective RNA target.

Methods

Results

Conclusion