Interaction of Nucleolin with an Evolutionarily Conserved Pre-ribosomal RNA Sequence Is Required for the Assembly of the Primary Processing Complex

Hervé Ginisty,Guillaume Serin,Laurence Ghisolfi-Nieto,Benoit Roger,Virginie Libante,François Amalric,Philippe Bouvet

doi:10.1074/jbc.m002350200

Abstract

The first processing event of the precursor ribosomal RNA (pre-rRNA) takes place within the 5' external transcribed spacer. This primary processing requires conserved cis-acting RNA sequence downstream from the cleavage site and several nucleic acids (small nucleolar RNAs) and proteins trans-acting factors including nucleolin, a major nucleolar protein. The specific interaction of nucleolin with the pre-rRNA is required for processing in vitro. Xenopus laevis and hamster nucleolin interact with the same pre-rRNA site and stimulate the processing activity of a mouse cell extract. A highly conserved 11-nucleotide sequence located 5-6 nucleotides after the processing site is required for the interaction of nucleolin and processing. In vitro selection experiments with nucleolin have identified an RNA sequence that contains the UCGA motif present in the 11-nucleotide conserved sequence. The interaction of nucleolin with pre-rRNA is required for the formation of an active processing complex. Our findings demonstrate that nucleolin is a key factor for the assembly and maturation of pre-ribosomal ribonucleoparticles.

Highlights

Ribosome biogenesis is a complex process that involves the transcription of a large pre-rRNA1 precursor, its maturation, and assembly with ribosomal proteins [1, 2]
A labeled RNA corresponding to nucleotides ϩ541 to ϩ1250 (RNA541/1250) from the mouse pre-rRNA was incubated in mouse S100 extract in presence of increasing amount of X. laevis or CHO nucleolin and subjected to an UV cross-linking experiment
These results suggest that the activation of the processing is more efficient when nucleolin interacts with the pre-rRNA substrate first. This first step might be required for an efficient recruitment of the processing complex. In these experiments we show that the interaction of nucleolin with an evolutionary conserved RNA sequence (ECM) in the pre-rRNA 5Ј-external transcribed spacers (ETS) is required for processing and formation of a specific complex

Summary

Introduction

Ribosome biogenesis is a complex process that involves the transcription of a large pre-rRNA1 precursor, its maturation, and assembly with ribosomal proteins [1, 2]. The different factors assemble in a large 20 S complex [18] that could be visualized at the terminal ends of nascent pre-rRNA (terminal balls) observed on Miller’s Christmas tree by electron microscopy [19, 20] The formation of this complex seems necessary but not sufficient for processing [20, 21]. The 200 nt that follow this motif are 80% conserved between mice and humans [5, 8, 23] and stimulate the processing [21] This RNA sequence can be the targets of several RNA-binding proteins or small nucleolar RNAs for the recognition of the cleavage site. Specific interactions of nucleolin with the pre-RNA substrate and with other cellular components like the U3snoRNP are required for the processing

Methods

Results

Conclusion