Placeholder factors in ribosome biogenesis: please, pave my way

Abstract

The synthesis of cytoplasmic eukaryotic ribosomes is an extraordinarily energy-demanding cellular activity that occurs progressively from the nucleolus to the cytoplasm. In the nucleolus, precursor rRNAs associate with a myriad of trans-acting factors and some ribosomal proteins to form pre-ribosomal particles. These factors include snoRNPs, nucleases, ATPases, GTPases, RNA helicases, and a vast list of proteins with no predicted enzymatic activity. Their coordinate activity orchestrates in a spatiotemporal manner the modification and processing of precursor rRNAs, the rearrangement reactions required for the formation of productive RNA folding intermediates, the ordered assembly of the ribosomal proteins, and the export of pre-ribosomal particles to the cytoplasm; thus, providing speed, directionality and accuracy to the overall process of formation of translation-competent ribosomes. Here, we review a particular class of trans-acting factors known as "placeholders". Placeholder factors temporarily bind selected ribosomal sites until these have achieved a structural context that is appropriate for exchanging the placeholder with another site-specific binding factor. By this strategy, placeholders sterically prevent premature recruitment of subsequently binding factors, premature formation of structures, avoid possible folding traps, and act as molecular clocks that supervise the correct progression of pre-ribosomal particles into functional ribosomal subunits. We summarize the current understanding of those factors that delay the assembly of distinct ribosomal proteins or subsequently bind key sites in pre-ribosomal particles. We also discuss recurrent examples of RNA-protein and protein-protein mimicry between rRNAs and/or factors, which have clear functional implications for the ribosome biogenesis pathway.

Keywords: RNA mimicry; ribosomal proteins; ribosome assembly; trans-acting factors; yeast.

Figure 1. Figure 1: Mrt4 and Mex67 act as placeholder factors for the P0 r-protein.

(A) Position of Mrt4 (red) in the early pre-60S r-particles purified with Nog2-TAP (PDB ID: 3JCT; 40). (B) Mex67-binding sites at the P0 neighbourhood (green), identified by CRAC , have been highlighted in the late/cytoplasmic Nmd3-TAP pre-60S r-particle (PDB ID: 5H4P; . (C) Position of P0 (red) in the mature 60S r-subunit (PDB ID: 3U5I, 3U5H; 44). Particles are viewed from the subunit interface slightly turned to the left. For orientation, the positions of the 5S rRNA (yellow), the L9 r-protein (royal blue) and the above CRAC sites of Mex67 (green) have been highlighted in the three structures. The ITS2 foot has also labelled in A. Note that some of the CRAC sites of Mex67 overlap with Mrt4 and P0 in A and C, respectively. The rest of rRNAs are coloured in pale blue and the rest of r-proteins and/or factors in light cornflower blue. Images were generated using the UCSF Chimera program (www.cgl.ucsf.edu/chimera).

Figure 2. Figure 2: Rlp24 functions as a placeholder factor for the L24 r-protein.

(A) Position of Rlp24 (red) in the early pre-60S r-particles purified with Nog2-TAP (PDB ID: 3JCT; 40). (B) Position of L24 r-protein (red) in the mature 60S r-subunit (PDB ID: 3U5I, 3U5H; 44). Particles are viewed from the subunit interface. For orientation, the positions of the 5S rRNA (yellow) and the L23 r-protein (royal blue) have been highlighted. The ITS2 foot has also labelled in A. Note that the last ca. 50 amino acids from the C-terminal part of Rlp24 and ca. 20 amino acids from the C-terminal end of L24 could not be modelled in the respective structures. The rest of rRNAs are coloured in pale blue and the rest of r-proteins and/or factors in light cornflower blue.

Figure 3. Figure 3: Rlp7 is not the placeholder factor for the assembly of L7 r-protein.

(A) Position of Rlp7 (red) and L7 (purple) in the early pre-60S r-particles purified with Nog2-TAP (PDB ID: 3JCT; 40). (B) Position of L7 r-protein (purple) in the mature 60S r-subunit (PDB ID: 3U5I, 3U5H; 44). Particles are viewed from the solvent side. For orientation, the positions of the 5S rRNA (yellow), 5.8S rRNA (gold) and the L9 r-protein (royal blue) have been highlighted. The ITS2 foot has also labelled in A. Note that L7 is found at its final assembly position within Nog2-TAP pre-60S r-particles. The first ca. 20 amino acids of both proteins could not be modelled in either structure. The rest of rRNAs are coloured in pale blue and the rest of r-proteins and/or factors in light cornflower blue.

Figure 4. Figure 4: Imp3 is not the placeholder factor for the assembly of S9 r-protein.

(A) Position of Imp3 (red) and S9 (medium blue) in the 90S pre-ribosomal particle, also known as the SSU processome (PDB ID: 5TZS; 93). (B) Position of S9 r-protein (royal blue) in the mature 40S r-subunit (PDB ID: 3U5B, 3U5C; 46). Mature 40S r-subunit is seen from the A-site view, and the 90S pre-ribosomal particles has been consequently oriented from a similar position regarding the nascent 40S r-subunit. The positions of the U3 snoRNP (yellow) and that of the S4 r-protein (cyan) have been highlighted. The 5' domain of 18S rRNA has been coloured in dark grey, the central domain in medium grey, and the rest of 18S rRNAs in pale blue; r-proteins and/or factors have been coloured in light cornflower blue.

Figure 5. Figure 5: Interactions at the interface side of cytoplasmic pre-60S ribosomal particles.

(A) The Nmd3-binding sites, identified by CRAC at 25S rRNA helices H38, H69-69 and H89-90 , have been highlighted in dark grey in a reconstituted 60S r-subunit (PDB ID:5T62; 114). The CRAC sites common for Nmd3, Dbp10 and Nug1 are labelled in green. (B) Position of Nog2 (red), Nsa2 (blue), Nog1 (gold) and Nug1 (pink) in the early pre-60S r-particle purified with Nog2-TAP (PDB ID: 3JCT; 40). Note that only a very small portion of Nug1 has been resolved in this particle. (C) Position of Nmd3 and Lsg1 in a reconstituted 60S r-subunit (PDB ID:5T62; 114). Note that only the region of Nmd3 comprised between residues 46 and 388 of 518 in total is shown. The N-terminal end of Nmd3 approaches to Tif6 while the C-terminal end contact L1. In A-C, the position of Tif6 (purple) is shown. The locations of the 5S rRNA (yellow) and that of the L23 (gold) and L9 r-proteins (royal blue) have also been highlighted. (D) Position of SBDS (dark gold) in the 60S r-subunit. The structure of a pre-60S r-particle from Dyctiostelium discoideum containing Tif6 and reconstituted with human SBDS and EFL1 (PDB ID: 5ANB; 116) was superimposed on the structure of yeast 60S r-subunit (PDB ID: 5APN; 68); then, all common proteins from D. discoideium were removed from the model. L9 (royal blue), L40 (navy blue), L10 (violet), L23 (gold) and 5S rRNA (yellow) were highlighted. Note that SBDS is shown in its open conformation state. (E) As D, but additionally showing EFL1 (red) on top of Tif6. In all figures, the Nmd3-CRAC sites shown in A (dark grey) were also highlighted, the rest of rRNAs are coloured in pale blue and the rest of r-proteins and/or factors in light cornflower blue.

Figure 6. Figure 6: Interactions at the polypeptide exit tunnel of pre-60S ribosomal particles.

(A) Position of Nog1 (red) and Arx1 (dark gold) in the early pre-60S r-particle purified with Nog2-TAP (PDB ID: 3JCT; 40). Note that the C-terminal part of Nog1 enters the tunnel. The location of Mrt4 (cyan) is also shown. (B) Position of Rei1 (red) and Arx1 (dark gold) in a reconstituted 60S r-subunit (PDB ID: 5APN; 68). Note that only the middle part (141-261) and the C-terminal end (300-393) of the protein is visualised. As above, the C-terminal part of Rei1 enters the tunnel. The location of P0 (cyan) is also shown. (C) Position of Reh1 (red) in the late/cytoplasmic pre-60S r-particles purified with Nmd3-TAP (PDB ID: 5H4P; 43). Note that only the region of the protein occluding the tunnel could be visualised (amino acids 377-432 of 432). Particles are viewed from the solvent side. For orientation, the locations of L5 (yellow) and L39 (blue) have been highlighted. The rest of rRNAs are coloured in pale blue and the rest of r-proteins and/or factors in light cornflower blue.

Figure 7. Figure 7: The helix H84 in the 60S r-subunit, the symportin Syo1 and the p53 regulator MDM2 share the same binding site on L11 r-protein.

(A) Structure of the 5S RNP as is assembled in the mature 60S r-subunit bound to 25S rRNA (nucleotides 2651-2750 comprising helices H83, H84, H85 and H86). (B) Interaction of a specific region of Syo1, called Syo1-HS (amino acids 328-384), with L11 in the context of the 5S RNP. (C) Structure of the 5S RNP bound to a distinct fragment of MDM2 (amino acids 293-334). The fragment of 25S rRNA is coloured in yellow, that of Syo1 in light gold, and that of MDM2 in dark gold; 5S rRNA in highlighted in red, L5 in cyan and L11 in blue. PDB ID: The 5S RNP bound to 25S rRNA was taken from 3U5I and 3U5H ; Syo1-HS fragment was extracted from 5AFF after superimposing the structure shown here with that present in the PDB file 4GMN ; MDM2 fragment was taken from 4XXP after superimposing the structure shown in this file with that of L11 shown in A.