Repression of RNA Polymerase I Transcription by Nucleolin Is Independent of the RNA Sequence That Is Transcribed

Abstract

Nucleolin is one of the most abundant non-ribosomal proteins of the nucleolus. Several studies in vitro have shown that nucleolin is involved in several steps of ribosome biogenesis, including the regulation of rDNA transcription, rRNA processing, and ribosome assembly. However, the different steps of ribosome biogenesis are highly coordinated, and therefore it is not clear to what extent nucleolin is involved in each of these steps. It has been proposed that the interaction of nucleolin with the rDNA sequence and with nascent pre-rRNA leads to the blocking of RNA polymerase I (RNA pol I) transcription. To test this model and to get molecular insights into the role of nucleolin in RNA pol I transcription, we studied the function of nucleolin in Xenopus oocytes. We show that injection of a 2-4-fold excess of Xenopus or hamster nucleolin in stage VI Xenopus oocytes reduces the accumulation of 40 S pre-rRNA 3-fold, whereas transcription by RNA polymerase II and III is not affected. Direct analysis of rDNA transcription units by electron microscopy reveals that the number of polymerase complexes/ rDNA unit is drastically reduced in the presence of increased amounts of nucleolin and corresponds to the level of reduction of 40 S pre-rRNA. Transcription from DNA templates containing various combinations of RNA polymerase I or II promoters in fusion with rDNA or CAT sequences was analyzed in the presence of elevated amounts of nucleolin. It was shown that nucleolin leads to transcription repression from a minimal polymerase I promoter, independently of the nature of the RNA sequence that is transcribed. Therefore, we propose that nucleolin affects RNA pol I transcription by acting directly on the transcription machinery or on the rDNA promoter sequences and not, as previously thought, through interaction with the nascent pre-rRNA.The synthesis of functional ribosomes is a major task for the cell. Ribosomal gene transcription can account for as much as 40% of all cellular transcription and ribosomal RNA for about 80% of the RNA content of living cells (1). The different steps of ribosome biogenesis take place in a subcompartment of the nucleus called the nucleolus (2-4). The localization of the different steps of ribosome biogenesis in a single nuclear compartment probably allows an efficient coordination and regulation of ribosome assembly. The formation of mature ribosomes is one of the most complex assembly of ribonucleoparticles involving the interaction of four different RNAs and about 80 ribosomal proteins (5). In addition, several nucleolar non-ribosomal proteins are required for this process (6 -8). An ordered interaction of ribosomal and non-ribosomal proteins with pre-rRNA is probably required for the formation of functional ribosomes. The molecular details of this highly integrated process are still largely unknown.The non-ribosomal proteins fibrillarin and nucleolin as well as some ribosomal proteins have been detected on nascent prerRNA (9 -11) suggesting that they interact with the pre-rRNA during transcrip...

Characterization of AtNUC-L1 Reveals a Central Role of Nucleolin in Nucleolus Organization and Silencing of AtNUC-L2 Gene in Arabidopsis

“…However, it is probably that both transcription and processing of prerRNA are affected in Atnuc-L1. Indeed, in vitro assays and microinjection experiments in X. laevis oocytes showed a role of nucleolin in both transcription of pre-rRNA (Roger et al, 2002) and in the first step of pre-rRNA processing (Ginisty et al, 1998). Moreover, our results also show that AtNUC-L1 binds the rDNA promoter in vivo; consequently, we expect that the absence of AtNUC-L1 in mutant plants might also affect rDNA condensation and gene transcription.…”

“…However, mutations that abolish nucleolin gene expression have not yet been reported in a higher organism. Thus, most of our knowledge on the function of this protein in higher eukaryotic cells comes from in vitro studies using acellular systems, microinjection experiments, or overexpression of a nucleolin transgene in Xenopus oocytes (Yanagida et al, 2001;Roger et al, 2002Roger et al, , 2003. Only two reports have described the effect of reduction of nucleolin in higher organisms.…”

A Plant snoRNP Complex Containing snoRNAs, Fibrillarin, and Nucleolin-Like Proteins Is Competent for both rRNA Gene Binding and Pre-rRNA Processing In Vitro

“…Overexpression of nucleolin in Xenopus oocytes repressed RNA Pol I transcription and affected the packaging of nascent pre-rRNA and the formation of 40S subunits (44). Interestingly, the effect of nucleolin was dependent on RNA Pol I transcription (43).…”

Cell Cycle-controlled Interaction of Nucleolin with the Retinoblastoma Protein and Cancerous Cell Transformation

“…A role of nucleolin in activation as well as repression of ribosomal RNA synthesis has been described (1,66). Thus far, it has not been completely elucidated how the intracellular distribution of nucleolin is controlled.…”