Bop1 Is a Mouse WD40 Repeat Nucleolar Protein Involved in 28S and 5.8S rRNA Processing and 60S Ribosome Biogenesis

Abstract

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...

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