Nucleolin is an abundant protein of the nucleolus. Nucleolar proteins structurally related to nucleolin are found in organisms ranging from yeast to plants and mammals. The association of several structural domains in nucleolin allows the interaction of nucleolin with different proteins and RNA sequences. Nucleolin has been implicated in chromatin structure, rDNA transcription, rRNA maturation, ribosome assembly and nucleo-cytoplasmic transport. Studies of nucleolin over the last 25 years have revealed a fascinating role for nucleolin in ribosome biogenesis. The involvement of nucleolin at multiple steps of this biosynthetic pathway suggests that it could play a key role in this highly integrated process.

Structure and functions of nucleolin

The first processing step of precursor ribosomal RNA (pre-rRNA) involves a cleavage within the 5' external transcribed spacer. This processing requires sequences downstream of the cleavage site which are perfectly conserved among human, mouse and Xenopus and also several small nucleolar RNAs (snoRNAs): U3, U14, U17 and E3. In this study, we show that nucleolin, one of the major RNA-binding proteins of the nucleolus, is involved in the early cleavage of pre-rRNA. Nucleolin interacts with the pre-rRNA substrate, and we demonstrate that this interaction is required for the processing reaction in vitro. Furthermore, we show that nucleolin interacts with the U3 snoRNP. Increased levels of nucleolin, in the presence of the U3 snoRNA, activate the processing activity of a S100 cell extract. Our results suggest that the interaction of nucleolin with the pre-rRNA substrate might be a limiting step in the primary processing reaction. Nucleolin is the first identified metazoan proteinaceous factor that interacts directly with the rRNA substrate and that is required for the processing reaction. Potential roles for nucleolin in the primary processing reaction and in ribosome biogenesis are discussed.

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Nucleolin functions in the first step of ribosomal RNA processing.

Nucleolin, a major nucleolar phosphoprotein, is presumed to function in rDNA transcription, rRNA packaging and ribosome assembly. Its primary sequence was highly conserved during evolution and suggests a multi-domain structure. To identify structural elements required for nuclear uptake and nucleolar accumulation of nucleolin, we used site-directed mutagenesis to introduce point- and deletion-mutations into a chicken nucleolin cDNA. Following transient expression in mammalian cells, the intracellular distribution of the corresponding wild-type and mutant proteins was determined by indirect immunofluorescence microscopy. We found that nucleolin contains a functional nuclear localization signal (KRKKEMANKSAPEAKKKK) that conforms exactly to the consensus proposed recently for a bipartite signal (Robbins, J., Dilworth, S.M., Laskey, R.A. and Dingwall, C. (1991) Cell 64, 615-623). Concerning nucleolar localization, we found that the N-terminal 250 amino acids of nucleolin are dispensible, but deletion of either the centrally located RNA-binding motifs (the RNP domain) or the glycine/arginine-rich C terminus (the GR domain) resulted in an exclusively nucleoplasmic distribution. Although both of these latter domains were required for correct subcellular localization of nucleolin, they were not sufficient to target non-nucleolar proteins to the nucleolus. From these results we conclude that nucleolin does not contain a single, linear nucleolar targeting signal. Instead, we propose that the protein uses a bipartite NLS to enter the nucleus and then accumulates within the nucleolus by virtue of binding to other nucleolar components (probably rRNA) via its RNP and GR domains.

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Protein localization to the nucleolus: a search for targeting domains in nucleolin

Nucleolin is a major protein of exponentially growing eukaryotic cells where it is present in abundance at the heart of the nucleolus. It is highly conserved during evolution. Nucleolin contains a specific bipartite nuclear localization signal sequence and possesses a number of unusual structural features. It has unique tripartite structure and each domain performs a specific function by interacting with DNA or RNA or proteins. Nucleolin exhibits intrinsic self-cleaving, DNA helicase, RNA helicase and DNA-dependent ATPase activities. Nucleolin also acts as a sequence-specific RNA binding protein, an autoantigen, and as the component of a B cell specific transcription factor. Its phosphorylation by cdc2, CK2, and PKC-zeta modulate some of its activities. This multifunctional protein has been implicated to be involved directly or indirectly in many metabolic processes such as ribosome biogenesis (which includes rDNA transcription, pre-rRNA synthesis, rRNA processing, ribosomal assembly and maturation), cytokinesis, nucleogenesis, cell proliferation and growth, cytoplasmic-nucleolar transport of ribosomal components, transcriptional repression, replication, signal transduction, inducing chromatin decondensation and many more (see text). In plants it is developmentally, cell-cycle, and light regulated. The regulation of all these functions of a single protein seems to be a challenging puzzle.

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Nucleolin: A Multifunctional Major Nucleolar Phosphoprotein

The poliovirus 3′ noncoding region (3′NCR) is involved in the efficient synthesis of viral negative-stranded RNA molecules. A strong interaction between a 105-kDa host protein and the wild-type 3′NCR, but not with a replication-defective mutant 3′NCR, was detected. This 105-kDa protein was identified as nucleolin which predominantly resides in the nucleolus and has been proposed to function in the folding of rRNA precursor molecules. A functional role for nucleolin in viral genome amplification was examined in a cell-free extract which has been shown to support the assembly of infectious virus from virion RNA. At early times of viral gene expression, extracts depleted of nucleolin produced less infectious virus than extracts depleted of fibrillarin, another resident of the nucleolus, indicating a functional role of nucleolin in the early stages of the viral life cycle in this in vitro system. Immunofluorescence analysis of uninfected and infected cells showed a nucleocytoplasmic relocalization of nucleolin, but not of fibrillarin, in poliovirus-infected cells. Relocalization of nucleolin was not simply a consequence of virally induced inhibition of translation or transcription, because inhibitors of translation or transcription did not induce nucleolar-cytoplasmic relocalization of nucleolin. These findings suggest a novel virus-induced mechanism by which certain nucleolar proteins are selectively redistributed in infected cells.

M.L. Escande-Géraud

Papers

Correlation between rDNA transcription and distribution of a 100 kD nucleolar protein in CHO cells.