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, 14 (9), 1918-29

The Role of Human Ribosomal Proteins in the Maturation of rRNA and Ribosome Production

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The Role of Human Ribosomal Proteins in the Maturation of rRNA and Ribosome Production

Sara Robledo et al. RNA.

Abstract

Production of ribosomes is a fundamental process that occurs in all dividing cells. It is a complex process consisting of the coordinated synthesis and assembly of four ribosomal RNAs (rRNA) with about 80 ribosomal proteins (r-proteins) involving more than 150 nonribosomal proteins and other factors. Diamond Blackfan anemia (DBA) is an inherited red cell aplasia caused by mutations in one of several r-proteins. How defects in r-proteins, essential for proliferation in all cells, lead to a human disease with a specific defect in red cell development is unknown. Here, we investigated the role of r-proteins in ribosome biogenesis in order to find out whether those mutated in DBA have any similarities. We depleted HeLa cells using siRNA for several individual r-proteins of the small (RPS6, RPS7, RPS15, RPS16, RPS17, RPS19, RPS24, RPS25, RPS28) or large subunit (RPL5, RPL7, RPL11, RPL14, RPL26, RPL35a) and studied the effect on rRNA processing and ribosome production. Depleting r-proteins in one of the subunits caused, with a few exceptions, a decrease in all r-proteins of the same subunit and a decrease in the corresponding subunit, fully assembled ribosomes, and polysomes. R-protein depletion, with a few exceptions, led to the accumulation of specific rRNA precursors, highlighting their individual roles in rRNA processing. Depletion of r-proteins mutated in DBA always compromised ribosome biogenesis while affecting either subunit and disturbing rRNA processing at different levels, indicating that the rate of ribosome production rather than a specific step in ribosome biogenesis is critical in patients with DBA.

Figures

FIGURE 1.
FIGURE 1.
Efficient depletion of r-protein mRNA in HeLa using siRNA. Cells were transfected with scrambled negative control siRNA or siRNAs targeting the RPS6, RPS7, RPS15, RPS16, RPS17, RPS19, RPS24, RPS25, RPS28, RPL7, RPL14, RL26, or RPL35a mRNAs. After 72 h, total RNA was extracted and subjected to Northern blotting analysis. Membranes were hybridized with cDNA probes for one of each r-protein; mouse β-actin was used as a loading control. The percentage decrease in the targeted mRNA by the siRNA is indicated (right side for each panel). (A) Efficient depletion of targeted small subunit (RPS) mRNAs. A single experiment where equivalent amounts of RNAs were analyzed on three separate blots. Note RNA from the RPS6 depletion experiment is overloaded in this experiment. (B) Efficient depletion of targeted large subunit (RPL) mRNAs.
FIGURE 2.
FIGURE 2.
Knocking down ribosomal proteins decreases the levels of other ribosomal proteins of the same subunit. Western blot analysis of r-protein levels after siRNA treatment. Total cellular protein was extracted 72 h after treatment with siRNA specific to each target or a scrambled sequence. (A) Western blotting was performed to examine the levels of RPS6, RPS12, RPS16, RPS19, RPS24, RPS25, RPL26; GAPDH was used as a loading control. Knocking down all RPS proteins analyzed decreased the levels of other RPS proteins, with the exception of RPS25. RPS25 protein levels were decreased by treatment with siRNA against RPS25, although the levels of the other r-proteins of the small subunit examined did not change. The levels of RPL proteins did not decrease. (B) Western blot analysis of RPL7, RPL26, and RPS19 protein levels after depletion of RPL proteins; GAPDH was used as a loading control. Knocking down all RPL proteins analyzed decreased the levels of other RPL proteins. The levels of r-proteins of the small subunit did not decrease, except in the RPL26 knockdown where a slight reduction of RPS19 levels was observed.
FIGURE 3.
FIGURE 3.
Depletion of r-proteins impairs the synthesis of new ribosomes. After 72 h of treatment with siRNA, cells were treated with cycloheximide, lysed, and the cytoplasmic fraction was layered over 10%–45% sucrose gradients. Mock or scramble treated cells show a typical profile of free 40S, free 60S, 80S, and polysomes. Cells depleted for RPS6, RPS7, RPS15, RPS16, RPS17, RPS19, RPS24, and RPS28 have significantly decreased levels of free 40S and 80S, with a dramatic increase in free 60S. The polysome levels are decreased. RPS25 depleted cells show an intermediate phenotype, with a slight decrease in free 40S, an increase in free 60S, and partial decrease in 80S. Depleting cells of RPL5, RPL7, RPL11, RPL14, and RPL35a show an increase in free 40S subunits, a substantial decrease in free 60S subunits, and a decrease in the 80S. Polysome profiles also demonstrate the presence of halfmers and a decrease in polysomes, particularly the larger polysomes. Depletion of RPL26 also decreased the free 60S subunit and the 80S and produced halfmer polysomes, although the free 40S levels did not increase as dramatically as was seen with other RPL knockdowns.
FIGURE 4.
FIGURE 4.
Ribosomal proteins of the small subunit control distinct rRNA processing steps important for the maturation of the 18S rRNA. (A) Schematic diagram of pre-rRNA processing pathways in HeLa cells. (Inset) Structure of the primary 47S rRNA transcript containing two external transcriber spacers at its 5′ and 3′ ends (5′ETS and 3′ETS, respectively), as well two internal transcriber spacers (ITS1 and ITS2). (Arrows) Position of major processing sites (0–4). The 47S pre-rRNA is processed through intermediate precursors designated according to their relative sedimentation coefficients (S) to mature 18S, 28S, and 5.8S rRNAs. In HeLa cells, 45S pre-rRNA can be processed by two alternative pathways. Pathway A is initiated with the removal of 5′-ETS by cleavage at site 1, while pathway B starts with cleavage in ITS1, probably at site 2 (Hadjiolova et al. 1993; Rouquette et al. 2005; Idol et al. 2007). The sequence locations of the probes used in Northern blot analysis are shown as a line above the primary transcript scheme (bottom). (B) Northern hybridization of total RNA from HeLa cells after depletion of r-proteins of the small subunit. Cells were transfected with scrambled negative control siRNA or siRNAs targeting RPS6, RPS7, RPS15, RPS16, RPS17, RPS19, RPS24, RPS25, or RPS28 mRNAs. After 72 h, total RNA was extracted and subjected to Northern blotting analysis for pre-rRNA species. Membranes were hybridized with probes for the 5′ end of ITS1 (probe i, upper panel), 5′ ends of ITS2 (probe f, lower panel), or β-actin. (Left panel) Short exposure, (right panel) long exposure. Depletion of r-proteins of the small subunit block rRNA processing at specific steps. The sizes of pre-rRNA species are indicated between the left and right panels. (C) Northern hybridization of total (T), nuclear (N), and cytoplasmic (C) rRNA isolated from HeLa cells after depletion of r-proteins of RPS15 and RPS25. Cells were transfected with scrambled negative control siRNA or siRNAs targeting RPS15 and RPS25 mRNAs. Membranes were hybridized with probes for the 5′ end of ITS1 (probe i).
FIGURE 5.
FIGURE 5.
Impaired 18S rRNA synthesis in HeLa cells depleted of r-proteins of the small subunit. Cells were transfected with scrambled negative control siRNA or siRNAs targeting RPS6, RPS7, RPS15, RPS16, RPS17, RPS19, RPS24, RPS25, or RPS28 mRNAs. After 72 h, cells were pulsed with L-[methyl3H] methionine and then chased at 0, 30, 60, and 120 min. Total rRNA was extracted after labeling and analyzed by fluorography.
FIGURE 6.
FIGURE 6.
Ribosomal proteins of the large subunit control distinct rRNA processing steps, in particular, those important for the maturation of the 28S rRNA and 5.8S rRNA. (A) Northern hybridization of total RNA from HeLa cells after depletion of r-proteins of the large subunit. Cells were transfected with scrambled negative control siRNA or siRNAs targeting RPL5, RPL7, RPL11, RPL14, RPL26, or RPL35a mRNAs. After 72 h, total RNA was extracted and subjected to Northern blotting analysis for pre-rRNA species. Membranes were hybridized with probes for the 5′ end of ITS1 (probe i, upper panel) and 5′ end of ITS2 (probe f, lower panel). Pre-rRNA products after hybridization with probe i (upper panel) and probe f (lower panel). (Left panel) Short exposure, (right panel) long exposure. The sizes of pre-rRNA species are indicated between the left and right panels. This short run is shown to emphasize the characteristic difference in 5.8S and 12S accumulation. (B) Analysis of the rRNA synthesis of 18S and 28S in HeLa cells depleted of r-proteins of the large subunit by pulse-chase analysis with L-[methyl3H] methionine labeling. Cells were transfected with scrambled negative control siRNA or siRNAs targeting RPL5, RPL7, RPL11, RPL14, RL26, or RPL35a mRNAs. After 72 h, cells were pulsed with L-[methyl3H] methionine and then chased at 0, 30, 60, and 120 min. Total rRNA was extracted after labeling and analyzed by fluorography.

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