Ribosome Biogenesis in Yeast

Nascent 60S ribosomal subunits enter the free pool bound by Nmd3p

“…GST-Nmd3p and free GST were expressed in wild-type yeast (CH1305) and purified from total-cell extracts by glutathione sepharose column chromatography as directed by the manufacturer (Pharmacia)+ 60S subunits were purified from strain CH1305 as described (Raué et al+, 1991)+ Protein binding reactions were carried out as follows+ GST-Nmd3p (2 mg) or free GST (2 mg) were incubated with 0+2 A 260 U of purified 60S subunits in 50 mL of joining buffer (10 mM Tris-HCl, pH 7+5, 50 mM KCl, 8 mM MgCl 2 , 6 mM b-mercaptoethanol) for 30 min at 30 8C+ Reactions were brought to 0+5 mL by the addition of joining buffer and then 20 mL of BSA-coated glutathione sepharose 4B beads were added+ Samples were incubated with rocking for 1 h+ Beads were pelleted by brief centrifugation and washed five times in joining buffer+ Bound proteins were then eluted by the addition of buffer containing 50 mM glutathione+…”

“…In eukaryotic cells, ribosomal subunits are assembled in the nucleolus (Kressler et al+, 1999b;Venema & Tollervey, 1999) and are subsequently exported from the nucleus as preassembled complexes (Warner, 1989;Raué & Planta, 1991;Venema & Tollervey, 1999)+ Nuclear export requires nuclear export signals (NESs) on the cargo molecules and recognition of these signals by receptor proteins that then mediate export via interaction with the nuclear pore complex (Mattaj & Englmeier, 1998;Stutz & Rosbash, 1998;Görlich & Kutay, 1999;Nakielny & Dreyfuss, 1999;Strässer & Hurt, 1999)+ Our previous work on Nmd3p from yeast showed that Nmd3p is an essential protein required for a late step in biogenesis of the large (60S) ribosomal subunit+ This was most clearly demonstrated in a temperaturesensitive nmd3-4 mutant, which, at nonpermissive temperature, processed rRNAs with normal kinetics, but the resulting nascent 25S rRNA was extremely unstable (t 1/2 ϭ 4 min)+ The stability of mature subunits made before the temperature shift did not appear to be affected (Ho & Johnson, 1999)+ Because the processing and assembly of ribosomal subunits are tightly coordinated (Warner, 1989(Warner, , 1999, we interpreted the instability of 25S rRNA to indicate a failure in the 60S biogenesis pathway after subunit assembly+ In related experiments, the overexpression of a truncated Nmd3p led to inhibition of 60S subunit biogenesis )+ Although Nmd3p is predominantly a cytoplasmic protein and associated with free 60S subunits (subunits not associated with 40S subunits) (Ho & Johnson, 1999), we have recently shown that Nmd3p shuttles and that it is required for nuclear export of the 60S subunit (Ho et al+, 2000)+ Indeed, deletion of the NES of Nmd3p traps both Nmd3p and 60S subunits in the nucleus+ Thus Nmd3p may provide the NES for export of the 60S subunit+ 60S subunits entering the cytoplasm require the loading of several additional ribosomal proteins, including the small acidic proteins P1 and P2 and possibly three other proteins, including the exchangeable protein Rpl10p (Zinker & Warner, 1976;Eisinger et al+, 1997)+ Kinetic analysis in yeast of the appearance of 60S subunits in the cytoplasm and their incorporation into actively translating polysomes indicates that there is a pool of newly formed 60S subunits not yet in polysomes, suggesting the existence of a slow cyto-plasmic maturation step (Warner, 1971)+ Human ribosome biogenesis in HeLa cells also displays a slow cytoplasmic 60S step (Warner, 1966)+ The ...…”

Nuclear and nucleolar targeting of human ribosomal protein S6.

“…cDNA Fragments Most ribosomal proteins have to accumulate in the nucleolus to take part in the assembly of the ribosomal subunits (Hadjiolov, 1985;Warner, 1990;Raue and Planta, 1991). Knowing the nucleolar targeting properties of the construct pPbxS6-3 ( Figures 1B and 2C), we decided to further analyze the sequence requirements for nucleolar accumulation of ribosomal protein S6.…”

“…The biogenesis of functional ribosomal subunits in the nucleolus is a highly complex process requiring the exact control and regulation of a large number of molecular interactions (Hadjiolov, 1985;Warner, 1990;Raue and Planta, 1991). The complexity of ribosome formation in eucaryotic cells is due to the involvement of all three RNA-polymerases in the synthesis of ribosomal components.…”

Nop58p is a common component of the box C+D snoRNPs that is required for snoRNA stability

“…Eukaryotic ribosomal RNAs (rRNAs) are synthesized from precursor rRNAs (pre-rRNAs) through a complex processing pathway (Fig+ 1; see Eichler & Craig, 1994;Venema & Tollervey, 1995;Sollner-Webb et al+, 1996;Tollervey, 1996 for recent reviews)+ While these processing reactions take place, the pre-rRNAs are covalently modified on both the sugar residues (29-O-methylation) and bases (pseudouridine formation and base methylation) (Maden, 1990;Maden & Hughes, 1997) and assemble with the ribosomal proteins into ribonucleoprotein (RNP) particles (Warner, 1989;Raué & Planta, 1991)+ Most of these steps occur in the nucleolus, a specialized subnuclear compartment (Reeder, 1990;Hernandez-Verdun, 1991;Mélèse & Xue, 1995)+ Eukaryotic nucleoli contain a large number of small, metabolically stable RNAs known collectively as the small nucleolar RNAs (snoRNAs) (reviewed in Fournier & Maxwell, 1993;Bachellerie et al+, 1995;Maxwell & Fournier, 1995); some 150 snoRNA species are predicted to be present in human cells+ Recently, it has become apparent that these snoRNAs fall into two classes that are structurally and functionally distinct (Balakin et al+, 1996;Ganot et al+, 1997b;Tollervey & Kiss, 1997;reviewed in Lafontaine & Tollervey, 1998)+ These are designated the box CϩD and the box HϩACA snoRNAs after conserved sequence elements that are believed to be sites of RNA-protein interactions+ The only exception is the RNA component of the endonuclease RNase MRP, which is related to RNase P (Forster & Altman, 1990;Lygerou et al+, 1994; reviewed in Morrissey & Tollervey, 1995)+ Within each major family of snoRNAs, two functionally distinct groups can be discerned+ A small number of snoRNA species-the box HϩACA snoRNA snR30 and the box CϩD snoRNAs U3 and U14-are required for cleavage of the pre-rRNA at the early processing sites, A 0 , A 1 , and A 2 (Fig+ 1; Li et al+, 1990;Hughes & Ares, 1991;Morrissey & Tollervey, 1993)+ Since these cleavages are required for synthesis of the 18S rRNA, this group of snoRNAs are essential for viability+ In contrast, the vast majority of snoRNAs function as guide RNAs for the covalent modification of the pre-...…”