We have conducted a genetic screen in order to identify ribosomal proteins of Saccharomyces cerevisiae involved in nuclear export of the small subunit precursors. This has led us to distinguish Rps15p as a protein dispensable for maturation of the pre-40S particles, but whose assembly into the pre-ribosomes is a prerequisite to their nuclear exit. Upon depletion of Rps15p, 20S pre-rRNA is released from the nucleolus and retained in the nucleus, without alteration of the pre-rRNA early cleavages. In contrast, Rps18p, which contacts Rps15p in the small subunit, is required upstream for pre-rRNA processing at site A2. Most pre-40S specific factors are correctly associated with the intermediate particles accumulating in the nucleus upon Rps15p depletion, except the late-binding proteins Tsr1p and Rio2p. Here we show that these two proteins are dispensable for nuclear exit; instead, they participate in 20S pre-rRNA processing in the cytoplasm. We conclude that, during the final maturation steps in the nucleus, incorporation of the ribosomal protein Rps15p is specifically required to render the pre-40S particles competent for translocation to the cytoplasm.

Approximately 25% of cases of Diamond Blackfan anemia, a severe hypoplastic anemia, are linked to heterozygous mutations in the gene encoding ribosomal protein S19 that result in haploinsufficiency for this protein. Here we show that deletion of either of the two genes encoding Rps19 in yeast severely affects the production of 40 S ribosomal subunits. Rps19 is an essential protein that is strictly required for maturation of the 3-end of 18 S rRNA. Depletion of Rps19 results in the accumulation of aberrant pre-40 S particles retained in the nucleus that fail to associate with pre-ribosomal factors involved in late maturation steps, including Enp1, Tsr1, and Rio2. When introduced in yeast Rps19, amino acid substitutions found in Diamond Blackfan anemia patients induce defects in the processing of the pre-rRNA similar to those observed in cells underexpressing Rps19. These results uncover a pivotal role of Rps19 in the assembly and maturation of the pre-40 S particles and demonstrate for the first time the effect of Diamond Blackfan anemiaassociated mutations on the function of Rps19, strongly connecting the pathology to ribosome biogenesis. Diamond Blackfan anemia (DBA)5 is a severe hypoplastic anemia that generally presents early in infancy (1, 2). Other clinical features of DBA are heterogeneous with some patients presenting craniofacial abnormalities, growth failure, predisposition to cancer, and other congenital abnormalities. Most cases of DBA arise spontaneously, with only a small proportion exhibiting familial transmission typically showing autosomal dominant inheritance. Approximately 25% of the DBA cases have been linked to mutations in the gene encoding ribosomal protein S19 (RPS19), and in these cases haploinsufficiency for this ribosomal protein gives rise to the disease (3, 4). The remaining cases are of unknown etiology.The Rps19 protein is a component of the small ribosomal subunit, and as such, defects in some aspect of ribosome structure, function, or synthesis may be an underlying cause of DBA. The human Rps19 protein belongs to a family of ribosomal proteins restricted to eukaryotes and archea. Rps19 does not have a homolog in the eubacterial ribosome where the properties of individual ribosomal proteins have been most extensively studied. As such, little is known regarding the function of members of the eukaryotic Rps19 family. In prokaryotes, ribosomal proteins play critical roles in ribosomal assembly through their interactions with each other and rRNA (5). These interactions promote both steps in rRNA processing important for subunit maturation and the formation of active sites in mature subunits necessary for ribosome function. The precise functions of the ribosomal proteins in the production of the subunits in eukaryotes are poorly characterized, which calls for a more detailed investigation of the role played by Rps19 in ribosome synthesis and function.The yeast Saccharomyces cerevisiae has proven to be an outstanding system to investigate factors involved in eukaryotic ribosome synthesis...

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.