Functions of ribosomal proteins in assembly of eukaryotic ribosomes in vivo

Abstract

The proteome of cells is synthesized by ribosomes, complex ribonucleoproteins that in eukaryotes contain 79-80 proteins and four ribosomal RNAs (rRNAs) more than 5,400 nucleotides long. How these molecules assemble together and how their assembly is regulated in concert with the growth and proliferation of cells remain important unanswered questions. Here, we review recently emerging principles to understand how eukaryotic ribosomal proteins drive ribosome assembly in vivo. Most ribosomal proteins assemble with rRNA cotranscriptionally; their association with nascent particles is strengthened as assembly proceeds. Each subunit is assembled hierarchically by sequential stabilization of their subdomains. The active sites of both subunits are constructed last, perhaps to prevent premature engagement of immature ribosomes with active subunits. Late-assembly intermediates undergo quality-control checks for proper function. Mutations in ribosomal proteins that affect mostly late steps lead to ribosomopathies, diseases that include a spectrum of cell type-specific disorders that often transition from hypoproliferative to hyperproliferative growth.

Keywords: 40S ribosomal subunits; 60S ribosomal subunits; RNA–protein interactions; pre-rRNA processing; rRNA folding; ribosome assembly.

Figure 1

Crystal structure of (a,b) the small subunit (SSU) and (c,d) the large subunit (LSU) from Saccharomyces cerevisiae at 3.0-Å resolution. (a,c) The subunit interface of the SSU and LSU, respectively. (b,d) The solvent-exposed surface of the SSU and LSU, respectively. Abbreviations: CP, central protuberance. GAC, GTPase-activation center. The crystal structure is adapted from Protein Data Bank codes 3U5B, 3U5C, 3U5D, and 3U5E.

Figure 2

Cotranscriptional precursor rRNA (pre-rRNA) processing in Saccharomyces cerevisiae. (a) As RNA polymerase I transcribes a ribosomal DNA (rDNA) repeat, nascent pre-rRNAs are cleaved cotranscriptionally at the A₂ site, releasing a 43S preribosome containing 20S pre-rRNA. The 66S preribosome containing 27SA₂ pre-rRNA is released upon completion of transcription. The pre-rRNA processing sites are indicated along the rDNA gene, and the external and internal transcribed spacer sequences are indicated on the nascent transcript. (b) The pre-rRNAs then undergo a series of exo- and endonucleolytic cleavages to remove the spacer sequences, finally liberating mature 18S, 5.8S, and 25S rRNAs. Not shown is the flanking 5S gene, transcribed in the opposite direction.

Figure 3

Correlation of function and location of the small subunit (SSU) and large subunit (LSU) r-proteins of Saccharomyces cerevisiae. Early-acting (yellow), middle-acting (blue), and late-acting (red) r-proteins are mapped onto the crystal structure. (a,c) The subunit interface of the SSU and LSU, respectively. (b,d) The solvent-exposed surface of the SSU and LSU, respectively. Ribosomal RNA (rRNA) and r-proteins are shown in cartoon and surface representation, respectively. Abbreviations: CP, central protuberance; GAC, GTPase-activation center. The crystal structure is adapted from Protein Data bank codes 3U5D and 3U5E.

Figure 4

(a) Cytoplasmic steps of 40S incorporation. (i) A 40S assembly intermediate containing seven stably bound assembly factors accumulates in the cytoplasm at steady state. Phosphorylation of Ltv1 by the kinase Hrr25 releases Ltv1 and Enp1, allowing for (ii) repositioning of S3 and incorporation of S10 at the messenger RNA (mRNA) entry channel (76). (iii) Release of Ltv1 allows for eIF5B-dependent joining of the 60S subunit to form 80S-like ribosomes. (iv) Before Fap7 acts on 80S-like ribosomes, Rio2 is released, and independently, Asc1 joins. (v) Dim1 is released (J. Trepreau & K. Karbstein, unpublished data) before (vi) Nob1-dependent 18S formation and Tsr1 release. (vii) Tsr1 release allows for binding of Dom34/Rli1 to dissociate 80S-like ribosomes. (viii) Exchange of Pno1 for S26 occurs in polysomes (97). The nascent 40S subunit is shown in light gray, the mature 60S subunit in dark gray, r-proteins in magenta, stably bound assembly factors in yellow, transiently bound assembly factors/translation factors in green, and mRNA in blue. S3 and S10 are in lighter shades to indicate their location on the solvent side of the molecule. (b) Cytoplasmic maturation of pre-60S ribosomal particles. Pre-60S ribosomal particles that arrive in the cytoplasm contain only a few stably bound export adaptors (Arx1–Alb1, Mex67–Mtr2, Nmd3, Bud20) and assembly factors (Mtr4, Nog1, Rlp24, Tif6) that are sequentially released to enable assembly of the remaining r-proteins. Note that the exact order of some steps (e.g., release of Mex67–Mtr2, assembly of L29) has still not been properly addressed. (i) The first step is the release of Rlp24, Nog1, and Bud20 by the ATPase Drg1, which then permits the assembly of L24 and the recruitment of Rei1. (ii) Rei1, together with the J protein Jjj1 and the HSP70 ATPase Ssa, enables the release of Arx1–Alb1, located near the polypeptide exit tunnel. Thus, this functional ribosomal site is inactive until the release of Arx1–Alb1. (iii) Then, or in parallel, Yvh1 is required for the removal of Mrt4, which is replaced in the pre-60S particles by the stalk r-protein P0. The stalk is required for recruitment of translation elongation factors (eEFs); thus, pre-60S particles lacking P0 are inactive. (iv) Pre-60S particles containing P0 are able to recruit the GTPase Efl1, which is closely related to eEF2. Efl1, together with Sdo1, facilitates the release of Tif6 from pre-60S ribosomal subunits (r-subunits). Tif6 inhibits r-subunit joining, thus preventing pre-60S particles from prematurely engaging in translation. (v) The release of Tif6 leads to activation of the GTPase Lsg1 to release the export adaptor Nmd3. Assembly of L40 and L10, aided by the chaperone Sqt1, is also required for the release of Nmd3. Nmd3 binds to the joining surface of the 60S r-subunit, thus also impeding joining with 40S r-subunits. Release of Nmd3 allows the 60S r-subunits to gain translation competence. Finally, acidic r-proteins P1 and P2 assemble to the r stalk at the moment when the mature 60S r-subunit is joined to the 40S r-subunit and committed to translation.