Bop1 is a novel nucleolar protein involved in rRNA processing and ribosome assembly. We have previously shown that expression of Bop1⌬, an amino-terminally truncated Bop1 that acts as a dominant negative mutant in mouse cells, results in inhibition of 28S and 5.8S rRNA formation and deficiency of newly synthesized 60S ribosomal subunits (Z. Strezoska, D. G. Pestov, and L. F. Lau, Mol. Cell. Biol. 20:5516-5528, 2000). Perturbation of Bop1 activities by Bop1⌬ also induces a powerful yet reversible cell cycle arrest in 3T3 fibroblasts. In the present study, we show that asynchronously growing cells are arrested by Bop1⌬ in a highly concerted fashion in the G 1 phase. Kinase activities of the G 1 -specific Cdk2 and Cdk4 complexes were downregulated in cells expressing Bop1⌬, whereas levels of the Cdk inhibitors p21 and p27 were concomitantly increased. The cells also displayed lack of hyperphosphorylation of retinoblastoma protein (pRb) and decreased expression of cyclin A, indicating their inability to progress through the restriction point. Inactivation of functional p53 abrogated this Bop1⌬-induced cell cycle arrest but did not restore normal rRNA processing. These findings show that deficiencies in ribosome synthesis can be uncoupled from cell cycle arrest and reveal a new role for the p53 pathway as a mediator of the signaling link between ribosome biogenesis and the cell cycle. We propose that aberrant rRNA processing and/or ribosome biogenesis may cause "nucleolar stress," leading to cell cycle arrest in a p53-dependent manner.Proliferating cells can delay or block cell cycle transitions in response to a variety of extracellular regulatory signals as well as to perturbations in intracellular processes. Several types of stress, such as DNA damage, defects in replication and chromosome segregation, and accumulation of misfolded proteins in the endoplasmic reticulum are now known to elicit checkpoint responses that prevent progression through the cell cycle (16,25,69). These responses are often altered in neoplastic cells, suggesting that the regulatory mechanisms involved play important roles in tumor development (24).In a previous study, we applied a genetic selection procedure to search for sequences in a cDNA library that can cause reversible arrest of the cell cycle (45). One cDNA clone (Bop1⌬) that induced a particularly strong inhibition of DNA synthesis in NIH 3T3 fibroblasts encoded an amino-terminally truncated form of a novel WD40 repeat protein, named Bop1 (block of proliferation). Expression of Bop1⌬ interfered with the functions of the endogenous Bop1 in a dominant manner, which likely accounted for the strong growth-inhibitory potential of this clone.Subsequent studies revealed that Bop1 was predominantly localized to the nucleolus and cofractionated with preribosomal particles (58). Bop1⌬ exhibited a similar localization but lacked some of the critical functions of the wild-type protein, leading to a dominant negative phenotype. Expression of this mutant form of Bop1 in LAP3 cells completely bloc...

We have identified and characterized a novel mouse protein, Bop1, which contains WD40 repeats and is highly conserved through evolution. bop1 is ubiquitously expressed in all mouse tissues examined and is upregulated during mid-G 1 in serum-stimulated fibroblasts. Immunofluorescence analysis shows that Bop1 is localized predominantly to the nucleolus. In sucrose density gradients, Bop1 from nuclear extracts cosediments with the 50S-80S ribonucleoprotein particles that contain the 32S rRNA precursor. RNase A treatment disrupts these particles and releases Bop1 into a low-molecular-weight fraction. A mutant form of Bop1, Bop1⌬, which lacks 231 amino acids in the N-terminus, is colocalized with wild-type Bop1 in the nucleolus and in ribonucleoprotein complexes. Expression of Bop1⌬ leads to cell growth arrest in the G 1 phase and results in a specific inhibition of the synthesis of the 28S and 5.8S rRNAs without affecting 18S rRNA formation. Pulse-chase analyses show that Bop1⌬ expression results in a partial inhibition in the conversion of the 36S to the 32S pre-rRNA and a complete inhibition of the processing of the 32S pre-rRNA to form the mature 28S and 5.8S rRNAs. Concomitant with these defects in rRNA processing, expression of Bop1⌬ in mouse cells leads to a deficit in the cytosolic 60S ribosomal subunits. These studies thus identify Bop1 as a novel, nonribosomal mammalian protein that plays a key role in the formation of the mature 28S and 5.8S rRNAs and in the biogenesis of the 60S ribosomal subunit.Biogenesis of the eukaryotic ribosomes occurs in the nucleolus, a complex nuclear organelle that forms around the nucleolar organizer regions located in heterochromatic chromosomal sites containing multiple rRNA genes (69, 81). The organization of the nucleolus and the assembly of ribosomes are coupled to transcription of rDNA by RNA polymerase I, which synthesizes a large primary precursor transcript. This precursor transcript is then processed into the mature 18S, 5.8S, and 28S/25S rRNAs. These rRNAs are assembled into preribosomes with some 80 ribosomal proteins that are transported into the nucleus, and with the 5S rRNA, transcribed by RNA polymerase III outside of the nucleolus (28). A large number of small nucleolar RNAs (snoRNAs) and nonribosomal proteins are also recruited to the nucleolus to participate in the modification, processing, and assembly of the rRNAs and proteins into ribonucleoprotein (RNP) particles. These preribosomal RNP (pre-rRNP) particles mature into nearly complete ribosomal subunits prior to their export out of the nucleus.Upon synthesis, the primary precursor rRNA transcript is modified by ribose methylation and pseudouridine conversion and processed through a series of nucleolytic cleavages into the matured rRNAs (21) (see Fig. 6). In vertebrates, the arrangement of the 47S primary precursor transcript begins with a 5Ј external transcribed spacer (5Ј-ETS), followed by the 18S rRNA, internal transcribed spacer 1 (ITS1), 5.8S rRNA, internal transcribed spacer 2 (ITS2), 28S rRNA, and t...

Molecular mechanisms of mammalian ribosome biogenesis remain largely unexplored. Here we develop a series of transposon-derived dominant mutants of Pes1, the mouse homolog of the zebrafish Pescadillo and yeast Nop7p implicated in ribosome biogenesis and cell proliferation control. Six Pes1 mutants selected by their ability to reversibly arrest the cell cycle also impair maturation of the 28S and 5.8S rRNAs in mouse cells. We show that Pes1 physically interacts with the nucleolar protein Bop1, and both proteins direct common pre-rRNA processing steps. Interaction with Bop1 is essential for the efficient incorporation of Pes1 into nucleolar preribosomal complexes. Pes1 mutants defective for the interaction with Bop1 lose the ability to affect rRNA maturation and the cell cycle. These data show that coordinated action of Pes1 and Bop1 is necessary for the biogenesis of 60S ribosomal subunits.

Ribosome biogenesis requires multiple nuclease activities to process pre-rRNA transcripts into mature rRNA species and eliminate defective products of transcription and processing. We find that in mammalian cells, the 5′ exonuclease Xrn2 plays a major role in both maturation of rRNA and degradation of a variety of discarded pre-rRNA species. Precursors of 5.8S and 28S rRNAs containing 5′ extensions accumulate in mouse cells after siRNA-mediated knockdown of Xrn2, indicating similarity in the 5′-end maturation mechanisms between mammals and yeast. Strikingly, degradation of many aberrant pre-rRNA species, attributed mainly to 3′ exonucleases in yeast studies, occurs 5′ to 3′ in mammalian cells and is mediated by Xrn2. Furthermore, depletion of Xrn2 reveals pre-rRNAs derived by cleavage events that deviate from the main processing pathway. We propose that probing of pre-rRNA maturation intermediates by exonucleases serves the dual function of generating mature rRNAs and suppressing suboptimal processing paths during ribosome assembly.