Despite having high-resolution structures for eukaryotic large ribosomal subunits, it remained unclear how these ribonucleoprotein complexes are constructed in living cells. Nevertheless, knowing where ribosomal proteins interact with ribosomal RNA (rRNA) provides a strategic platform to investigate the connection between spatial and temporal aspects of 60S subunit biogenesis. We previously found that the function of individual yeast large subunit ribosomal proteins (RPLs) in precursor rRNA (pre-rRNA) processing correlates with their location in the structure of mature 60S subunits. This observation suggested that there is an order by which 60S subunits are formed. To test this model, we used proteomic approaches to assay changes in the levels of ribosomal proteins and assembly factors in preribosomes when RPLs functioning in early, middle, and late steps of pre-60S assembly are depleted. Our results demonstrate that structural domains of eukaryotic 60S ribosomal subunits are formed in a hierarchical fashion. Assembly begins at the convex solvent side, followed by the polypeptide exit tunnel, the intersubunit side, and finally the central protuberance. This model provides an initial paradigm for the sequential assembly of eukaryotic 60S subunits. Our results reveal striking differences and similarities between assembly of bacterial and eukaryotic large ribosomal subunits, providing insights into how these RNA-protein particles evolved.

During the assembly process of ribosomal subunits, their structural components, the ribosomal RNAs (rRNAs) and the ribosomal proteins (r-proteins) have to join together in a highly dynamic and defined manner to enable the efficient formation of functional ribosomes. In this work, the assembly of large ribosomal subunit (LSU) r-proteins from the eukaryote S. cerevisiae was systematically investigated. Groups of LSU r-proteins with specific assembly characteristics were detected by comparing the protein composition of affinity purified early, middle, late or mature LSU (precursor) particles by semi-quantitative mass spectrometry. The impact of yeast LSU r-proteins rpL25, rpL2, rpL43, and rpL21 on the composition of intermediate to late nuclear LSU precursors was analyzed in more detail. Effects of these proteins on the assembly states of other r-proteins and on the transient LSU precursor association of several ribosome biogenesis factors, including Nog2, Rsa4 and Nop53, are discussed.

Defects in mitochondrial ribosomal proteins (MRPs) cause various diseases in humans. Because of the essential role of MRPs in synthesizing the essential subunits of oxidative phosphorylation (OXPHOS) complexes, identifying all of the protein components involved in the mitochondrial translational machinery is critical. Initially, we identified 79 MRPs; however, identifying MRPs with no clear homologs in bacteria and yeast mitochondria was challenging, due to limited availability of expressed sequence tags (ESTs) in the databases available at that time. With the improvement in genome sequencing and increased sensitivity of mass spectrometry (MS)-based technologies, we have established four previously known proteins as MRPs and have confirmed the identification of ICT1 (MRP58) as a ribosomal protein. The newly identified MRPs are MRPS37 (Coiled-coil-helix-coiled-coil-helix domain containing protein 1-CHCHD1), MRPS38 (Aurora kinase A interacting protein1, AURKAIP1), MRPS39 (Pentatricopeptide repeat-containing protein 3, PTCD3), in the small subunit and MRPL59 (CR-6 interacting factor 1, CRIF1) in the large subunit. Furthermore, we have demonstrated the essential roles of CHCHD1, AURKAIP1, and CRIF1in mitochondrial protein synthesis by siRNA knock-down studies, which had significant effects on the expression of mitochondrially encoded proteins.

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