Proteomic Analysis of Human Nop56p-associated Pre-ribosomal Ribonucleoprotein Complexes

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 prerRNA 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.The ribosome constitutes one of the most fundamental molecular machines in living cells. Given that protein synthesis is essential for cell growth, proliferation, and adaptation, ribosome biogenesis is intimately coupled to the needs of the cell. Ribosome biogenesis is efficiently coordinated in the nucleolus, a subnuclear compartment in eukaryotic cells. Each mature ribosome in the cytoplasm consists of the large subunit (60 S) and the small subunit (40 S) that together comprise over 80 ribosomal proteins organized within and around mature rRNAs. In mammalian cells, 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. During ribosome biogenesis, ribosomal DNA for 5.8 S, 18 S, and 28 S rRNAs is transcribed by RNA polymerase I into a large primary precursor (47 S) containing 5Ј-and 3Ј-external transcribed spacers (5Ј-ETS 1 and 3Ј-ETS) and two internal transcribed spacers (ITS1 and ITS2) (1, 2). Concomitant with the methylation and pseudouridylation of ribose moieties, the 47 S precursor is cleaved at specific sites to produce a series of characteristic intermediates that ultimately result in mature 5.8 S, 18 S, and 28 S rRNAs. Pre-5 S rRNA is transcribed by RNA polymerase III and processed independently of the other three rRNAs (3).During ribosome biogenesis, a multitude of ...

EBP1 is a nucleolar growth-regulating protein that is part of pre-ribosomal ribonucleoprotein complexes

“…It has been shown that nucleolin interacts with at least 60 different proteins, 40 of which are ribosomal (Yanagida et al, 2001), and it has been proposed that it acts as a shuttle for ribosomal proteins from the cytoplasm to the nucleolus and as a binding adapter of ribosomal proteins to rRNA, at a late stage of ribosome biogenesis. Recently, it has been found that preribosomal RNP complexes associate with human Nop56p, a component of the box C/D small RNP complexes involved in pre-rRNA maturation (Hayano et al, 2003). Proteins we have identified in EBP1 immunocomplexes were also identified in the Nop56p associated pre-ribosomal complexes.…”

The nucleolus: reviewing oldies to have new understandings

“…TCS results from loss-of-function of the TCOF1 gene products, treacle, a nucleolar protein sharing with several similarities of Nopp140. The TCS phenotype is also observed in Tcof1 heterozygous mice, suggesting that the correct dosage of treacle is essential for survival of cephalic neural crest cells, which contribute significantly to formation of branchial arches [60]. This case illustrates that the nucleolus Cell Research | www.cell-research.com…”

Silencing of RNA Helicase II/Guα Inhibits Mammalian Ribosomal RNA Production

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