M Phase Phosphoprotein 10 Is a Human U3 Small Nucleolar Ribonucleoprotein Component

Abstract

We have previously developed a novel technique for isolation of cDNAs encoding M phase phosphoproteins (MPPs). In the work described herein, we further characterize MPP10, one of 10 novel proteins that we identified, with regard to its potential nucleolar function. We show that by cell fractionation, almost all MPP10 was found in isolated nucleoli. By immunofluorescence, MPP10 colocalized with nucleolar fibrillarin and other known nucleolar proteins in interphase cells but was not detected in the coiled bodies stained for either fibrillarin or p80 coilin, a protein found only in the coiled body. When nucleoli were separated into fibrillar and granular domains by treatment with actinomycin D, almost all the MPP10 was found in the fibrillar caps, which contain proteins involved in rRNA processing. In early to middle M phase of the cell cycle, MPP10 colocalized with fibrillarin to chromosome surfaces. At telophase, MPP10 was found in cellular structures that resembled nucleolus-derived bodies and prenucleolar bodies. Some of these bodies lacked fibrillarin, a previously described component of nucleolus-derived bodies and prenucleolar bodies, however, and the bulk of MPP10 arrived at the nucleolus later than fibrillarin. To further examine the properties of MPP10, we immunoprecipitated it from cell sonicates. The resulting precipitates contained U3 small nucleolar RNA (snoRNA) but no significant amounts of other box C/D snoRNAs. This association of MPP10 with U3 snoRNA was stable to 400 mM salt and suggested that MPP10 is a component of the human U3 small nucleolar ribonucleoprotein.

Role of Pre-rRNA Base Pairing and 80S Complex Formation in Subnucleolar Localization of the U3 snoRNP

“…The U3 snoRNP is associated with a diverse number of proteins. Using antibodies specific for the common core box C/D proteins, as well as the U3-specific human (h) proteins hMpp10 and hU3-55K (13,44,47), we analyzed the association of these proteins with the wild-type and mutant U3 msl2 RNAs expressed in HEp-2 cells. Transfection experiments were performed as outlined above, and after 16 h of incubation extracts were prepared, immunoprecipitations were performed, and the coprecipitated RNAs were analyzed by Northern blot hybridization ( Fig.…”

Proteomic snapshot analyses of preribosomal ribonucleoprotein complexes formed at various stages of ribosome biogenesis in yeast and mammalian cells

“…Although, as in the case of yeast cells, the association of pre-rRNA with non-ribosomal proteins as RNP complexes persists during the maturation of 18S, 5.8S, and 28S rRNAs, and through assembly of ribosomal subunits in the nucleus (Piñol-Roma, 1999), only a few of the transacting proteins that are involved in ribosome biogenesis have been characterized in mammalian systems. The wellcharacterized trans-acting proteins are nucleolin (NCL), B23, fibrillarin (FIB), Mpp10, Bop1, and dyskerin (Ochs et al, 1985;Ginisty, Amalric, & Bouvet, 1998;Westendorf et al, 1998;Strezoska, Pestov, & Lau, 2000;Ruggero et al, 2003). Among those proteins, NCL, B23, and FIB have been actually found in the pre-rRNP complexes that were isolated from human HeLa cells by pull-down analysis with an anti-NCL antibody.…”

Sharing of mitotic pre-ribosomal particles between daughter cells

“…Based on their ultrastructure and composition, PNBs were initially thought to be a step prior to the formation of nucleoli. Because PNBs differ in their components and lifetimes, it was proposed that different types of PNBs exist that are targeted to reforming nucleoli with different kinetics (Savino et al, 1999(Savino et al, , 2001Westendorf et al, 1998). Beyond the dynamics of postmitotic formation of nucleoli, PNBs themselves are highly dynamic structures (Dundr et al, 2000;Muro et al, 2010).…”