The Assembly of Ribosomes in HeLa Cell Nucleoli

Abstract

The order in which ribosomal proteins become associated with nucleolar pre-rRNA was studied by measuring the kinetics of label accumulation in ribosomal proteins isolated from the cytoplasmic ribosomes, as well as by two-dimensional polyacrylamide gel electrophoresis of protein extracted from ribosome precursor particles. Tentative identifications of early-adding and late-adding groups of proteins have been made.The process by which ribosomes are assembled in growing HeLa cells has been studied by following the appearance of labeled rRNA [1] and the appearance of labeled ribosomal proteins [2,3] in cytoplasmic ribosomes at short times after addition of a labeled precursor to the medium. The rRNAs become labeled only after a lag period, which is about 20 min for the 1 8 4 rRNA and about 40 min for the 28-S rRNA. These lag periods are thought to represent the time between the transcription of the 45-S pre-rRNA and the appearance of the new ribosomal subunits in the cytoplasm (i.e. the minimum time required for rRNA transcription and processing, and for subunit assembly and transfer of the cytoplasm). When accumulation of labeled ribosomal proteins in the cytoplasmic ribosomes was examined, diflerent proteins were found to accumulate different amounts of radioactivity [2 -41. These results suggested that proteins enter the process of ribosome assembly at different times after the transcription of the 45-S pre-rRNA. This conclusion has received support from studies on the protein composition of ribosome precursor particles [5 -81. The experiments described below used a combination of kinetic measurements of label accumulation in the proteins of cytoplasmic ribosomes and direct enumeration of ribosomal precursor particle proteins on twodiinensional gels, to show that many proteins can be classified into early-adding and late-adding groups. MATERIALS AND METHODS Cell Growth und LhelingHeLa cells were grown in spinner culture flasks in Eagle's minimal essential medium supplemented with 10% calf serum (Gibco). The methionine in the medium used for the kinetic experiments was half that normally present (7.5 pg/ml instead of 15 pg/ml). Incorporation of [35S]methionine into acid-precipitable material (Fig. 3 ) was followed by resuspending 6.6 x lo7 exponentially growing HeLa cells in 25 ml Eagle's minimal essential medium supplemented with 5 calf serum. After 15 min, 50 pCi [35S]methionine was added. At the indicated times 1.0-ml samples were removed, filtered through Whatman GF/A filters, and immediately washed twice with unlabeled medium at 0°C. The filters were then heated in 5 % trichloroacetic acid (95 "C for 10 min) and vortexed in a small test-tube. After cooling to 0 'C, this solution and two 5 % trichloroacetic acid washes were filtered through a Millipore filter (0.45 pm pore size). The GF/A filter was combined with the Millipore filter in a scintialltion vial and counted by adding 0.6 ml water and 15 ml Aquasol (New England Nuclear).For the measurements of accumulation ratios (Table I), cells were grown for 3 da...

Reduced expression of the mouse ribosomal protein Rpl17 alters the diversity of mature ribosomes by enhancing production of shortened 5.8S rRNA

“…To address this possibility, we analyzed depletion of another essential r-protein, Rpl10 (uL16). Unlike Rpl17, Rpl10 is incorporated into nascent 60S subunits after their cytoplasmic export, as shown by previous studies in both mammalian cells (Auger-Buendia et al 1979;Lastick 1980) and yeast (Saveanu et al 2003;West et al 2005;Ohmayer et al 2013). Depletion of Rpl10 severely inhibited cell growth, as did depletion of Rpl17 (Fig.…”

“…Incorporation of r-proteins into preribosomes in vivo follows a broadly hierarchical order, according to which these proteins can be divided into several earlier-and later-acting assembly groups (Auger-Buendia et al 1979;Lastick 1980;Todorov et al 1983;O'Donohue et al 2010;Chen and Williamson 2013;Gamalinda et al 2014). Early-binding r-proteins promote subsequent assembly steps by facilitating structural rearrangements in pre-rRNA and enabling binding of assembly factors (Ferreira-Cerca et al 2007;Babiano and de la Cruz 2010;Jakob et al 2012;Ohmayer et al 2013).…”

Saccharomyces cerevisiae Ribosomal Protein L26 Is Not Essential for Ribosome Assembly and Function

“…Seminal work done in yeast and HeLa cells suggested that L26 assembles in the nucleus (54,55). Here, we show that functional GFP-tagged L26 proteins accumulate in the nucleus upon inhibition of nucleocytoplasmic export of pre-60S r-particles by overexpression of the dominant-negative Nmd3⌬100 protein.…”

Yeast Ribosomal Protein L40 Assembles Late into Precursor 60 S Ribosomes and Is Required for Their Cytoplasmic Maturation

“…In contrast, the single rpl40a⌬ and specially the rpl40b⌬ mutant were clearly much more sensitive to this drug than the wild-type strain; the IC 50 value obtained for the rpl40a⌬ and rpl40b⌬ mutants was at least 10-fold lower than that of the wild-type strain. Interestingly, this sensitivity is not simply related to a lower dosage of an r-protein because the IC 50 value of an rpl35b⌬ mutant is practically identical to that of the wild-type strain. Moreover, the effect of sordarin seems to be very specific because we could not have observed enhanced sensitivity or resistance to other translation antibiotics (anisomycin, cycloheximide, hygromycin B, paromomycin, and neomycin) for the above strains when compared with their isogenic wild-type counterpart.…”

“…One of the current challenges of the ribosome biogenesis field is defining the course of the assembly of r-proteins at a satisfactorily high resolution. A rough picture of this pathway has been obtained by pioneer work in yeast and HeLa cells (49,50). These studies indicate that most r-proteins are incorporated into nuclear pre-ribosomal particles either early or late in the process.…”