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

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.Ribosome biogenesis is a complex process that involves the transcription of a large pre-rRNA 1 precursor, its maturation, and assembly with ribosomal proteins (1, 2). Pre-rRNA undergoes multiple post-transcriptional nucleotide modification (3) and nucleolytic processing steps to yield the mature 18, 5.8, and 28 S rRNA species. The two first endonucleolytic cleavages occur in external transcribed spacers (ETS) and therefore do not lead to the formation of mature rRNA ends. The first cleavage also called early or primary processing occurs within the 5Ј-ETS (4 -6) is conserved from yeast (7) to human (8) and can be found at various positions within the 5Ј-ETS (5, 7-11).Despite its conservation, the role of this cleavage in ribosome biogenesis is still unknown, and only few factors involved in this process have been characterized. In yeast, the role of Rnt1p, an RNase III homologue remains unclear (12, 13). In higher eukaryotes, the major nucleolar protein nucleolin (15), an endonuclease (14) and several small nucleolar RNA are also involved (15, 16), but their exact function remains to be elucidated. In vitro UV cross-linking has identified a small number of proteins, including nucleolin, that interact with the RNA substrate (17, 18). 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 sequence and RNA structural motif required for the processing have been extensively studied in vitro (21,22). In mouse pre-rRNA, an evolutionary conserved 11-nt motif (ϩ655 to ϩ666) located just 5-6 nt downstream from the cleavage site is absolutely required for the processing (21) and for formation of the complex observed at the 5Ј end of nascent pre-rRNA (20). A 27-nt RNA (ϩ645 to ϩ...

Nucleolin and nucleophosmin: nucleolar proteins with multiple functions in DNA repair

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Regulation of DNA Replication after Heat Shock by Replication Protein A-Nucleolin Interactions

“…4A). Nucleolin is involved in ribosome biogenesis and is known to be required for the first step of pre-rRNA processing in which the 5Ј external transcribed spacer undergoes endonucleolytic cleavage (57,62,63). However, nucleolin is a multifunctional protein, has been found in a replication complex (64), and has been recently characterized as an RPA-interacting protein (58).…”

RNA-binding protein nucleolin in disease

“…14,27 Nucleolin also catalyzes the first processing step of pre-rRNA occurring in the 5' external transcribed spacer (5'-ETS) resulting in cleavage of the precursor transcript of rRNA. 28,29 The affinity of nucleolin for ribosomal proteins suggests that it may also help assemble ribosomal subunits in the nucleus, 15 and likely participates in the transport of these newly assembled ribosomal subunits into the cytoplasm. 30 Through its involvement in rDNA transcription, its association with nascent pre-rRNA, and its assembly and transport of ribosomal particles, nucleolin is believed to assist with every step of ribosome biosynthesis.…”