Abstract
Nop56p is a component of the box C/D small nucleolar ribonucleoprotein complexes that direct 2'-O-methylation of pre-rRNA during its maturation. Genetic analyses in yeast have shown that Nop56p plays important roles in the early steps of pre-rRNA processing. However, its precise function remains elusive, especially in higher eukaryotes. Here we describe the proteomic characterization of human Nop56p (hNop56p)-associated pre-ribosomal ribonucleoprotein complexes. Mass spectrometric analysis of purified pre-ribosomal ribonucleoprotein complexes identified 61 ribosomal proteins, 16 trans-acting factors probably involved in ribosome biogenesis, and 29 proteins whose function in ribosome biogenesis is unknown. Identification of pre-rRNA species within hNop56p-associated pre-ribosomal ribonucleoprotein complexes, coupled with the known functions of yeast orthologs of the probable trans-acting factors identified in human, demonstrated that hNop56p functions in the early to middle stages of 60 S subunit synthesis in human cells. Interestingly, the nucleolar phosphoprotein treacle, which is responsible for the craniofacial disorder associated with Treacher Collins syndrome, was found to be a constituent of hNop56p-associated pre-rRNP complexes. The association of hNop56p and treacle within the complexes was independent of rRNA integrity, indicating a direct interaction. In addition, the protein compositions of the treacle-associated and hNop56p-associated pre-ribosomal ribonucleoprotein complexes were very similar, suggesting functional similarities between these two complexes with respect to ribosome biogenesis in human cells.
Highlights
Nop56p is a component of the box C/D small nucleolar some biogenesis is intimately coupled to the needs of the cell
The large subunit is composed of ϳ50 ribosomal proteins and three species of rRNA (28 S, 5.8 S, and 5 S), whereas the small subunit consists of ϳ30 ribosomal proteins and 18 S rRNA
These results indicated that the purified complexes were ribonucleoproteins (RNPs) and that the association of the protein components was RNA-dependent
Summary
Materials—Human kidney cell line 293EBNA, Opti-MEM, and LipofectAMINE were obtained from Invitrogen. After washing the agarose five times with lysis buffer and once with 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, the bound complexes were eluted with 20 l of 50 mM Tris-HCl, pH 7.5, 150 mM NaCl containing 500 g/ml of the FLAG peptide. Ribonuclease Treatment of hNop56p-associated Complexes—Immunoprecipitated hNop56p-associated complexes (bound to anti-FLAG M2-agarose) were incubated in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl containing 10 g/ml RNase A for 10 min at 37 °C, washed twice with lysis buffer and once with 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and eluted with the FLAG peptide in the same manner described above. The cells were washed with PBS-T and incubated with Alexa Fluor 488conjugated anti-mouse IgG for 1 h at room temperature, followed by three washes with PBS-T. MS/MS spectra were acquired by data-dependent collision-induced dissociation, and the MS/MS data were analyzed using MASCOT software (Matrix Science, Wyndham Place, UK) for peptide assignment
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