The role of human ribosomal proteins in the maturation of rRNA and ribosome production

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.

Aberrant ribosome biogenesis activates c-Myc and ASK1 pathways resulting in p53-dependent G1 arrest

“…RpS3 knockdown raised a significant diminution of 18S rRNA (Figure 1e, left) and, in particular, an extensive accumulation of 30S and 32S pre-rRNA intermediates (Figure 1e, right). These results coincided with previous reports that characterized the depletion affects the rRNA processing by various siRNAs against ribosomal proteins (Gazda et al, 2008;Robledo et al, 2008). These data collectively indicate that rpS3 depletion actually induces ribosomal stress.…”

“…Moreover, the translational level of p53 in all siRNAtransfected cells did not change as previously shown in Supplementary Figure S1B (Supplementary Figure S5B). The results produced by RACK1 depletion can be interpreted as the same as the previous report showing that RACK1 is associated with ribosome in the cytoplasm at the final stage of the ribosome biogenesis, such as rpS15, or is not essential for the function of 40S small subunit such as rpS25 (Robledo et al, 2008). Finally, we checked ribosome profiles using a linear sucrose gradient as described in Materials and methods.…”

“…In previous reports, the downregulation of ribosomal proteins was shown to affect ribosome biogenesis, producing an abnormal ribosome profile (Idol et al, 2007;Robledo et al, 2008). Therefore, we analyzed the ribosome profile.…”

The NEDD8 inhibitor MLN4924 increases the size of the nucleolus and activates p53 through the ribosomal-Mdm2 pathway

“…5G). Similar defects in the 60S/40S ratio have been reported upon knockdown of large ribosome subunit proteins RPL7a, RPL11, RPL5, RPL14, RPL26, RPL35a 48,49,50 . The combination of the data, suggest that the activation of p53 by MLN4924 is not due to RNA Pol I inhibition but rather due to reorganisation of the nucleolar proteome that involves targeting RPs.…”

Evidence for a novel cytoplasmic processing event in ribosome maturation in the sea urchin Paracentrotus lividus

“…These ancillary molecules associate and dissociate from pre-ribosome and pre-ribosomal subunits during their movement from the nucleolus (the site of primary assembly) via the nucleus, to their final destination in the cytoplasm [1]. Different steps of pre-ribosome maturation have been elucidated in recent years (reviewed in [1][2][3][4][5][6][7][8][9][10][11]), but some intriguing aspects of these processes remain unclear. Among these, two important questions that remain are: (1) why the two ribosomal sub-units have different fates during maturation, and (2) why the different steps in the ribosomal particle maturation occur in different cellular compartments.…”