What Is the Role of RNA in Protein Synthesis?
Ribosome synthesis depends on nutrient availability, sensed by the target of rapamycin (TOR) signaling pathway in eukaryotes. TOR inactivation affects ribosome biogenesis at the level of rRNA gene transcription, expression of ribosomal proteins (r-proteins) and biogenesis factors, preribosome processing, and transport. Here, we demonstrate that upon TOR inactivation, levels of newly synthesized ribosomal subunits drop drastically before the integrity of the RNA polymerase I apparatus is severely impaired but in good correlation with a sharp decrease in r-protein production. Inhibition of translation by cycloheximide mimics the rRNA maturation defect observed immediately after TOR inactivation. Both cycloheximide addition and the depletion of individual r-proteins also reproduce TOR-dependent nucleolar entrapment of specific ribosomal precursor complexes. We suggest that shortage of newly synthesized r-proteins after short-term TOR inactivation is sufficient to explain most of the observed effects on ribosome production.
What role do ribosomes play in protein synthesis? - Answers
RNA Pol I transcription seems not to be the primary target of TOR inactivation, since we did not observe any significant qualitative or quantitative changes in the assembly of the RNA Pol I transcription machinery at the rDNA template after 15 min of rapamycin treatment (Fig. and ). Under these conditions RNA Pol I was still mobile, giving rise to nascent 35S pre-rRNA (Fig. and ), which does not, however, exclude the possibility that transcription elongation is partly affected (). Thus, rRNA synthesis likely continues after TOR inactivation while r-protein production is strongly impaired (Fig. ). This imbalance of structural components of the ribosome is then presumably adjusted by rapid degradation of misassembled, excess rRNA precursors. In support of this idea, in vivo depletion of individual r-proteins of the large or small ribosomal subunit leads frequently to a drastic relative accumulation of the corresponding other subunit both in yeast and in mammals (, , ). This strongly indicates that in eukaryotes misassembled ribosomal subunits are efficiently turned over (see reference for discussion).
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Most of these polypeptides migrated with the same velocity as r-proteins derived from an affinity-purified 80S ribosome (Fig. , bottom panels, compare lane 1 with lanes 2 and 8). We focused on the apparent level of neosynthesized proteins in two prominent bands (Fig. , bottom panels, asterisks), for which we further unambiguously identified r-proteins as major components by mass spectrometry (Table ). We found that production of these proteins was only moderately affected in the presence of 0.1 μg/ml cycloheximide, whereas they were no longer detectable in the rapamycin-treated sample (Fig. , lanes 8 to 10 in the bottom panels). 35S labeling of these proteins was similar, however, in cells treated with concentrations of 1 to 10 μg/ml cycloheximide (Fig. , bottom panels; compare lanes 9, 11, and 12). Thus, upon rapamycin treatment the production of r-proteins is specifically inhibited, which might be caused by the combination of downregulation of general translation () and the strong reduction in r-protein mRNA levels (, ).
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To directly correlate the reduction in r-protein production with the reduction in ribosome production, we performed pulse-chase experiments with [3H]uracil in the same strains used for the protein analysis. As expected, production of mature rRNAs was severely impaired upon rapamycin treatment (Fig. , compare lanes 2 and 4; quantification is shown in E). In the presence of 0.1 μg/ml cycloheximide, a concentration where total protein production (but not r-protein production) was reduced to the level observed after rapamycin treatment (Fig. ), robust 25S rRNA synthesis could still be observed (Fig. , lanes 2 and 6, and E). In contrast, the defect in 25S rRNA production was virtually identical in rapamycin-treated cells and cells incubated at concentrations of 1 to 10 μg/ml cycloheximide (Fig. , lanes 4, 8 and 10, and E), in which r-protein production was similarly affected (Fig. , lanes 9, 11, and 12 of the bottom panels). Thus, there is a very good correlation between r-protein production and rRNA synthesis.
Protein Synthesis Role Play - Illinois Institute of Technology
We speculated that limited r-protein production might be sufficient to explain nucleolar entrapment of preribosomal complexes observed after inhibition of translation and TOR signaling. We employed yeast strains with deletions of the chromosomal copies of RPS5 or RPL25 and conditionally expressing the respective wild-type allele under the control of the glucose repressible GAL1 promoter (, ). GAL1 promoter-dependent expression of RPS5 and RPL25 in these cells supports growth in galactose-containing medium. After transfer to glucose-containing medium—and thus repression of RPS5 or RPL25 expression—specific steps in nuclear maturation of either small (RPS5)- or large (RPL25)-ribosomal subunit precursors are strongly and selectively affected (, , , , ). We genetically modified these strains for constitutive expression of the Rrp12-GFP or the Nog1-GFP fusion protein from the respective genomic location. As a control, we included the corresponding wild-type strains expressing RPS5 and RPL25 from their endogenous loci and, in addition, the respective Rrp12-GFP or Nog1-GFP fusion protein. Exponentially growing cells in galactose-containing medium (YPG) were split into two parts. Half of the cells were transferred into glucose-containing medium (YPD), whereas the other half was again cultured in YPG medium. Incubation was continued for 90 min before live-cell imaging. In RPS5 and RPL25 wild-type strains no significant change in Rrp12-GFP or Nog1-GFP localization could be detected under the two different culture conditions (Fig. , compare panels YPG and YPD in columns 2 and 6). Wild-type-like localization of Rrp12-GFP or Nog1-GFP was also observed in the strains carrying RPS5 or RPL25 under the control of the conditional promoter cultured in YPG (Fig. , compare panel YPG in column 2 with column 4 and column 6 with column 8). In contrast, depletion of rpS5 and rpL25 in these strains cultured in YPD medium led to nucleolar entrapment of the respective GFP fusion proteins (Fig. , compare panels YPG with YPD in columns 4 and 8). Very similar results were obtained by depleting rpS14 in an Rrp12-GFP-expressing strain (data not shown).