Abstract
Ribosome biogenesis starts with transcription of the large ribosomal RNA precursor (47S pre-rRNA), which soon combines with numerous factors to form the 90S pre-ribosome in the nucleolus. Although the subsequent separation of the pre-90S particle into pre-40S and pre-60S particles is critical for the production process of mature small and large ribosomal subunits, its molecular mechanisms remain undetermined. Here, we present evidence that p32, fibrillarin (FBL), and Nop52 play key roles in this separation step. Mass-based analyses combined with immunoblotting showed that p32 associated with 155 proteins including 31 rRNA-processing factors (of which nine were components of small subunit processome, and six were those of RIX1 complex), 13 chromatin remodeling components, and six general transcription factors required for RNA polymerase III-mediated transcription. Of these, a late rRNA-processing factor Nop52 interacted directly with p32. Immunocytochemical analyses demonstrated that p32 colocalized with an early rRNA-processing factor FBL or Nop52 in the nucleolus and Cajal bodies, but was excluded from the nucleolus after actinomycin D treatment. p32 was present in the pre-ribosomal fractions prepared by cell fractionation or separated by ultracentrifugation of the nuclear extract. p32 also associated with pre-rRNAs including 47S/45S and 32S pre-rRNAs. Furthermore, knockdown of p32 with a small interfering RNA slowed the early processing from 47S/45S pre-rRNAs to 18S rRNA and 32S pre-rRNA. Finally, Nop52 was found to compete with FBL for binding to p32 probably in the nucleolus. Given the fact that FBL and Nop52 are associated with pre-ribosome particles distinctly different from each other, we suggest that p32 is a new rRNA maturation factor involved in the remodeling from pre-90S particles to pre-40S and pre-60S particles that requires the exchange of FBL for Nop52.
Highlights
We showed that 1) p32 associates with 31 proteins known or expected to be involved in rRNA processing, at least four of which are found to associate independently with p32; 2) p32 colocalizes with FBL or Nop52 in the nucleolus and is excluded from the nucleolus upon inhibition of transcription with actinomycin D; 3) p32 is present in the preribosomal fraction prepared by cell fractionation; 4) p32 coelutes with FBL or Nop52 in the pre-ribosomal fractions separated by ultracentrifugation; 5) p32 associates with prerRNAs, 6) p32 is present in the isolated Nop52-associated
Our hypothesis for p32 involvement in ribosome biogenesis is that p32 exchanges its binding partner, i.e. p32 binds Nop52 in place of FBL, in preribosomal particles in the nucleolus
This hypothesis is based on our results that 1) p32 association with Nop52 is independent of that with FBL; 2) p32 interacts with Nop52 without any cofactors, just as with FBL; 3) the association between FBL and p32 takes place in the nucleolus; 4) the association between Nop52 and p32 possibly occurs in the nucleolus, too; 5) p32 is required for the early processing of pre-rRNA from 47S/45S pre-rRNA to 18S rRNA and 32S pre-rRNA, 6) p32 associates with pre-rRNAs including 47S/ 45S and 32S pre-rRNAs, 7) p32 is present in Nop52-associated pre-60S ribosomal particles isolated from the nucleus; and 8) Nop52 competes with FBL for association with p32
Summary
Construction of Epitope-tagged Expression Plasmids—Reverse transcriptase (RT)-PCR was performed with the Super Script II kit (Invitrogen) according to the manufacturer’s instructions using total mRNA that was prepared from 293EBNA cells (Invitrogen). To prepare Nu, the nuclear pellet was sonicated in 500 l of lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM MgCl2, and 0.5% IGEPAL CA630) containing 1 mM PMSF and 20 U SUPERase1⁄7In (Ambion) three times for 20 s at 4 °C with a Bioruptor (Cosmo Bio) at the highest setting, and centrifuged for 15 min at 15,000 ϫ g. After removing the cytoplasm by the method described above, the pellet was sonicated in sonication buffer (25 mM Tris-HCl pH 8.0, 100 mM KCl, 1 mM NaF, 2 mM EDTA, 1 mM dithiothreitol, 0.05% IGEPAL CA-630, 1 mM PMSF, and 20 U SUPERase1⁄7In) three times for 20 s at 4 °C with a Bioruptor at the highest setting, and centrifuged for 15 min at 15,000 ϫ g. The transfected and intact cells were washed with PBS followed by permeabilization with PBS containing 0.5% (w/v) Triton
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
