The Experts below are selected from a list of 315 Experts worldwide ranked by ideXlab platform
Akiko Shimamura - One of the best experts on this subject based on the ideXlab platform.
Blood, 2011Co-Authors: Nicholas Burwick, Scott A Coats, Akiko ShimamuraAbstract:
Abstract 3438 Shwachman Diamond syndrome (SDS) is an autosomal recessive marrow failure syndrome with a predisposition to leukemia. Over 90% of SDS patients harbor biallelic mutations in the SBDS gene. SBDS has been implicated in several cellular functions including ribosome biogenesis and mitotic spindle stabilization. Deletion of SBDS orthologues in yeast results in a severe slow growth phenotype and depressed polysomes. Homozygous deletion of Sbds in murine models results in early embryonic lethality, while conditional deletion of Sbds in mouse liver demonstrates accumulation of 40S and 60S Subunits and halfmer formation consistent with impaired ribosome joining. SBDS facilitates the release of eIF6, a factor that prevents ribosome joining. The dramatic phenotypic and polysome changes noted in these experimental models were not observed in cells derived from SDS patients. SDS patient cells have only a mildly reduced growth rate compared to heatlhy controls, and polysome profiles do not demonstrate depressed polysomes or halfmer formation. Since complete abrogation of SBDS expression is lethal and biallelic null mutations in SBDS have not been reported, we examined the role of SBDS and eIF6 in SDS patients and human cell models. We first investigated whether ribosome Subunit homeostasis is impaired in SDS patient cells. We find that the 60S:40S Ribosomal Subunit ratio is consistently reduced in bone marrow stromal cells from SDS patients of different genotypes (n=4). This impairment in 60S:40S ratio is demonstrated in both SDS patient stromal cells and patient lymphoblasts. Stable lentiviral knockdown of SDS in normal marrow stromal cells recapitulates the reduction in 60S:40S ratio. SBDS and eIF6 co-sediment in polysome gradients of human SDS cells. This co-sedimentation is specific for the 60S Ribosomal Subunit. Since eIF6 has a role as an anti-joining factor, we next developed an in vitro assay to test for ribosome Subunit joining in human cells. In this assay, we validate that over-expression of eIF6 results in reduced ribosome joining, and eIF6 knockdown promotes ribosome joining. Moreover, we find that SDS patient stromal cells and patient lymphoblasts both demonstrate impaired ribosome Subunit joining, compared with healthy controls. Importantly, the addition of wild type SBDS or depletion of eIF6 improve ribosome joining in SDS patient cells. We demonstrate that the amino terminal sequences of SBDS are necessary but not sufficient for the association of SBDS with the 60S Ribosomal Subunit. Insertion of a patient-derived N-terminal SBDS point mutation also results in decreased association of SBDS with the 60S Ribosomal Subunit. These structure-function studies may help to inform genotype:phenotype correlations in SDS. The role of defective ribosome joining in promoting the SDS hematopoietic phenotype is of particular interest. Ongoing studies are interrogating the role of eIF6 modulation on the hematopoietic phenotype in SBDS- depleted cells. Insights garnered from these experiments will help inform the development of novel agents to improve the hematopoetic defect in human SDS. Disclosures: No relevant conflicts of interest to declare.
Blood, 2007Co-Authors: Karthik A Ganapathi, Karyn M Austin, Anusha P Dias, Robin Reed, Maggie Malsch, Akiko ShimamuraAbstract:
Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure, exocrine pancreatic dysfunction, and leukemia predisposition. Mutations in the SBDS gene are identified in most patients with SDS. SBDS encodes a highly conserved protein of unknown function. Data from SBDS orthologs suggest that SBDS may play a role in ribosome biogenesis or RNA processing. Human SBDS is enriched in the nucleolus, the major cellular site of ribosome biogenesis. Here we report that SBDS nucleolar localization is dependent on active rRNA transcription. Cells from patients with SDS or Diamond-Blackfan anemia are hypersensitive to low doses of actinomycin D, an inhibitor of rRNA transcription. The addition of wild-type SBDS complements the actinomycin D hypersensitivity of SDS patient cells. SBDS migrates together with the 60S large Ribosomal Subunit in sucrose gradients and coprecipitates with 28S Ribosomal RNA (rRNA). Loss of SBDS is not associated with a discrete block in rRNA maturation or with decreased levels of the 60S Ribosomal Subunit. SBDS forms a protein complex with nucleophosmin, a multifunctional protein implicated in ribosome biogenesis and leukemogenesis. Our studies support the addition of SDS to the growing list of human bone marrow failure syndromes involving the ribosome.
Blood, 2007Co-Authors: Stefan Karlsson, Karthik A Ganapathi, Karyn M Austin, Anusha P Dias, Robin Reed, Maggie Malsch, Akiko ShimamuraAbstract:
Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure, exocrine pancreatic dysfunction, and leukemia predisposition. Mutations in the SBDS gene are identified in most patients with SDS. SBDS encodes a highly conserved protein of unknown function. Data from SBDS orthologs suggest that SBDS may play a role in ribosome biogenesis or RNA processing. Human SBDS is enriched in the nucleolus, the major cellular site of ribosome biogenesis. Here we report that SBDS nucleolar localization is dependent on active rRNA transcription. Cells from patients with SDS or Diamond-Blackfan anemia are hypersensitive to low doses of actinomycin D, an Inhibitor of rRNA transcription. The addition of wild-type SBDS complements the actinomycin D hypersensitivity of SDS patient cells. SBDS migrates together with the 60S large Ribosomal Subunit in sucrose gradients and coprecipitates with 28S Ribosomal RNA (rRNA). Loss of SBDS is not associated with a discrete block in rRNA maturation or with decreased levels of the 60S Ribosomal Subunit. SBDS forms a protein complex with nucleophosmin, a multifunctional protein implicated in ribosome biogenesis and leukemogenesis. Our studies support the addition of SDS to the growing list of human bone marrow failure syndromes involving the ribosome.
John L Woolford - One of the best experts on this subject based on the ideXlab platform.
the assembly factor erb1 functions in multiple remodeling events during 60S Ribosomal Subunit assembly in s cerevisiaeNucleic Acids Research, 2017Co-Authors: Salini Konikkat, Stephanie Biedka, John L WoolfordAbstract:
A major gap in our understanding of ribosome assembly is knowing the precise function of each of the ∼200 assembly factors. The steps in Subunit assembly in which these factors participate have been examined for the most part by depleting each protein from cells. Depletion of the assembly factor Erb1 prevents stable assembly of seven other interdependent assembly factors with pre-60S Subunits, resulting in turnover of early preribosomes, before the ITS1 spacer can be removed from 27SA3 pre-rRNA. To investigate more specific functions of Erb1, we constructed eight internal deletions of 40-60 amino acid residues each, spanning the amino-terminal half of Erb1. The erb1Δ161-200 and erb1Δ201-245 deletion mutations block a later step than depletion of Erb1, namely cleavage of the C2 site that initiates removal of the ITS2 spacer. Two other remodeling events fail to occur in these erb1 mutants: association of twelve different assembly factors with domain V of 25S rRNA, including the neighborhood surrounding the peptidyl transferase center, and stable association of Ribosomal proteins with rRNA surrounding the polypeptide exit tunnel. This suggests that successful initiation of construction of these functional centers is a checkpoint for committing to spacer removal.
Biochemical Journal, 2017Co-Authors: Salini Konikkat, John L WoolfordAbstract:
Ribosome biogenesis requires the intertwined processes of folding, modification, and processing of Ribosomal RNA, together with binding of Ribosomal proteins. In eukaryotic cells, ribosome assembly begins in the nucleolus, continues in the nucleoplasm, and is not completed until after nascent particles are exported to the cytoplasm. The efficiency and fidelity of ribosome biogenesis are facilitated by >200 assembly factors and ∼76 different small nucleolar RNAs. The pathway is driven forward by numerous remodeling events to rearrange the ribonucleoprotein architecture of pre-ribosomes. Here, we describe principles of ribosome assembly that have emerged from recent studies of biogenesis of the large Ribosomal Subunit in the yeast Saccharomyces cerevisiae . We describe tools that have empowered investigations of ribosome biogenesis, and then summarize recent discoveries about each of the consecutive steps of Subunit assembly.
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Cristina Gutierrezvargas, Madhumitha Ramesh, Beril Tutuncuoglu, John L Woolford, Robert A Grassucci, Susan Madisonantenucci, Noel Espina, L Tong, Joachim FrankAbstract:
Abstract Ribosomes of trypanosomatids, a family of protozoan parasites causing debilitating human diseases, possess multiply fragmented rRNAs that together are analogous to 28S rRNA, unusually large rRNA expansion segments, and r-protein variations compared with other eukaryotic ribosomes. To investigate the architecture of the trypanosomatid ribosomes, we determined the 2.5-A structure of the Trypanosoma cruzi ribosome large Subunit by single-particle cryo-EM. Examination of this structure and comparative analysis of the yeast Ribosomal assembly pathway allowed us to develop a stepwise assembly model for the eight pieces of the large Subunit rRNAs and a number of ancillary “glue” proteins. This model can be applied to the characterization of Trypanosoma brucei and Leishmania spp. ribosomes as well. Together with other details, our atomic-level structure may provide a foundation for structure-based design of antitrypanosome drugs.
The N-terminal extension of yeast Ribosomal protein L8 is involved in two major remodeling events during late nuclear stages of 60S Ribosomal Subunit assembly.RNA, 2016Co-Authors: Beril Tutuncuoglu, Jelena Jakovljevic, Ning Gao, John L WoolfordAbstract:
Assaying effects on pre-rRNA processing and ribosome assembly upon depleting individual Ribosomal proteins (r-proteins) provided an initial paradigm for assembly of eukaryotic ribosomes in vivo-that each structural domain of Ribosomal Subunits assembles in a hierarchical fashion. However, two features suggest that a more complex pathway may exist: (i) Some r-proteins contain extensions that reach long distances across ribosomes to interact with multiple rRNA domains as well as with other r-proteins. (ii) Individual r-proteins may assemble in a stepwise fashion. For example, the globular domain of an r-protein might assemble separately from its extensions. Thus, these extensions might play roles in assembly that could not be revealed by depleting the entire protein. Here, we show that deleting or mutating extensions of r-proteins L7 (uL30) and L35 (uL29) from yeast reveal important roles in early and middle steps during 60S Ribosomal Subunit biogenesis. Detailed analysis of the N-terminal terminal extension of L8 (eL8) showed that it is necessary for late nuclear stages of 60S Subunit assembly involving two major remodeling events: removal of the ITS2 spacer; and reorganization of the central protuberance (CP) containing 5S rRNA and r-proteins L5 (uL18) and L11 (uL5). Mutations in the L8 extension block processing of 7S pre-rRNA, prevent release of assembly factors Rpf2 and Rrs1 from pre-ribosomes, which is required for rotation of the CP, and block association of Sda1, the Rix1 complex, and the Rea1 ATPase involved in late steps of remodeling.
Genes & Development, 2014Co-Authors: Michael Gamalinda, John L Woolford, Jelena Jakovljevic, Uli Ohmayer, Beril Kumcuoglu, Bertrade MbomAbstract:
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.
Alan J Warren - One of the best experts on this subject based on the ideXlab platform.
Advances in biological regulation, 2017Co-Authors: Alan J WarrenAbstract:
Mutations that target the ubiquitous process of ribosome assembly paradoxically cause diverse tissue-specific disorders (ribosomopathies) that are often associated with an increased risk of cancer. Ribosomes are the essential macromolecular machines that read the genetic code in all cells in all kingdoms of life. Following pre-assembly in the nucleus, precursors of the large 60S and small 40S Ribosomal Subunits are exported to the cytoplasm where the final steps in maturation are completed. Here, I review the recent insights into the conserved mechanisms of ribosome assembly that have come from functional characterisation of the genes mutated in human ribosomopathies. In particular, recent advances in cryo-electron microscopy, coupled with genetic, biochemical and prior structural data, have revealed that the SBDS protein that is deficient in the inherited leukaemia predisposition disorder Shwachman-Diamond syndrome couples the final step in cytoplasmic 60S Ribosomal Subunit maturation to a quality control assessment of the structural and functional integrity of the nascent particle. Thus, study of this fascinating disorder is providing remarkable insights into how the large Ribosomal Subunit is functionally activated in the cytoplasm to enter the actively translating pool of ribosomes.
Blood, 2016Co-Authors: Alan J WarrenAbstract:
Abstract The synthesis of new ribosomes is a fundamental conserved process in all cells. Ribosomes are pre-assembled in the nucleus and subsequently exported to the cytoplasm where they acquire functionality through a series of final maturation steps that include formation of the catalytic center, recruitment of the last remaining Ribosomal proteins and the removal of inhibitory assembly factors. Surprisingly, a number of key factors (SBDS, DNAJC21, RPL10 (uL16)) involved in late cytoplasmic maturation of the large (60S) Ribosomal Subunit are mutated in both inherited and sporadic forms of leukemia. In particular, biallelic mutations in the SBDS gene cause Shwachman-Diamond syndrome (SDS), a recessive bone marrow failure disorder with significant predisposition to acute myeloid leukemia. By using the latest advances in single-particle cryo-electron microscopy to elucidate the function of the SBDS protein, we have uncovered an elegant mechanism that couples final maturation of the 60S Subunit to a quality control assessment of the structural integrity of the active sites of the ribosome. Further molecular dissection of this pathway may inform novel therapeutic strategies for SDS and leukemia more generally. References: 1. Weis F, Giudice E, Churcher M,et al. Mechanism of eIF6 release from the nascent 60S Ribosomal Subunit. Nat Struct Mol Biol, (2015) Nov;22(11):914-9. 2. Wong CC, Traynor D, Basse N, et al. Defective ribosome assembly in Shwachman-Diamond syndrome. Plenary Paper, Blood. 2011 Oct 20;118(16):4305-12. 3. Finch AJ, Hilcenko C, Basse N, et al. Uncoupling of GTP hydrolysis from eIF6 release on the ribosome causes Shwachman-Diamond syndrome. Genes Dev (2011) 25: 917-929. 4. Menne TM, Goyenechea B, Sánchez-Puig N, et al. The Shwachman-Bodian-Diamond syndrome protein mediates translational activation of ribosomes in yeast. Nature Genetics (2007) 39: 486-95. Disclosures No relevant conflicts of interest to declare.
Nature Structural & Molecular Biology, 2015Co-Authors: Felix Weis, Emmanuel Giudice, Mark J. Churcher, Li Jin, Christine Hilcenko, Chi C. Wong, David Traynor, Robert R. Kay, Alan J WarrenAbstract:
SBDS protein (deficient in the inherited leukemia-predisposition disorder Shwachman-Diamond syndrome) and the GTPase EFL1 (an EF-G homolog) activate nascent 60S Ribosomal Subunits for translation by catalyzing eviction of the antiassociation factor eIF6 from nascent 60S Ribosomal Subunits. However, the mechanism is completely unknown. Here, we present cryo-EM structures of human SBDS and SBDS-EFL1 bound to Dictyostelium discoideum 60S Ribosomal Subunits with and without endogenous eIF6. SBDS assesses the integrity of the peptidyl (P) site, bridging uL16 (mutated in T-cell acute lymphoblastic leukemia) with uL11 at the P-stalk base and the sarcin-ricin loop. Upon EFL1 binding, SBDS is repositioned around helix 69, thus facilitating a conformational switch in EFL1 that displaces eIF6 by competing for an overlapping binding site on the 60S Ribosomal Subunit. Our data reveal the conserved mechanism of eIF6 release, which is corrupted in both inherited and sporadic leukemias.
Arlen W. Johnson - One of the best experts on this subject based on the ideXlab platform.
Journal of Cell Biology, 2012Co-Authors: Cyril Bussiere, Joachim Frank, Yaser Hashem, Sucheta Arora, Arlen W. JohnsonAbstract:
Eukaryotic ribosomes are preassembled in the nucleus and mature in the cytoplasm. Release of the antiassociation factor Tif6 by the translocase-like guanosine triphosphatase Efl1 is a critical late maturation step. In this paper, we show that a loop of Rpl10 that embraces the P-site transfer ribonucleic acid was required for release of Tif6, 90 A away. Mutations in this P-site loop blocked 60S maturation but were suppressed by mutations in Tif6 or Efl1. Molecular dynamics simulations of the mutant Efl1 proteins suggest that they promote a conformation change in Efl1 equivalent to changes that elongation factor G and eEF2 undergo during translocation. These results identify molecular signaling from the P-site to Tif6 via Efl1, suggesting that the integrity of the P-site is interrogated during maturation. We propose that Efl1 promotes a functional check of the integrity of the 60S Subunit before its first round of translation.
Characterization of the nuclear export adaptor protein Nmd3 in association with the 60S Ribosomal SubunitJournal of Cell Biology, 2010Co-Authors: Jayati Sengupta, Arlen W. Johnson, Cyril Bussiere, Jesper Pallesen, Matthew West, Joachim FrankAbstract:
The nucleocytoplasmic shuttling protein Nmd3 is an adaptor for export of the 60S Ribosomal Subunit from the nucleus. Nmd3 binds to nascent 60S Subunits in the nucleus and recruits the export receptor Crm1 to facilitate passage through the nuclear pore complex. In this study, we present a cryoelectron microscopy (cryo-EM) reconstruction of the 60S Subunit in complex with Nmd3 from Saccharomyces cerevisiae. The density corresponding to Nmd3 is directly visible in the cryo-EM map and is attached to the regions around helices 38, 69, and 95 of the 25S Ribosomal RNA (rRNA), the helix 95 region being adjacent to the protein Rpl10. We identify the interSubunit side of the large Subunit as the binding site for Nmd3. rRNA protection experiments corroborate the structural data. Furthermore, Nmd3 binding to 60S Subunits is blocked in 80S ribosomes, which is consistent with the assigned binding site on the Subunit joining face. This cryo-EM map is a first step toward a molecular understanding of the functional role and release mechanism of Nmd3.
Molecular Cell, 2010Co-Authors: Cyril Bussiere, Stefan Bresson, Edward M. Marcotte, Arlen W. JohnsonAbstract:
In eukaryotic cells the final maturation of ribosomes occurs in the cytoplasm, where trans-acting factors are removed and critical Ribosomal proteins are added for functionality. Here, we have carried out a comprehensive analysis of cytoplasmic maturation, ordering the known steps into a coherent pathway. Maturation is initiated by the ATPase Drg1. Downstream, assembly of the ribosome stalk is essential for the release of Tif6. The stalk recruits GTPases during translation. Because the GTPase Efl1, which is required for the release of Tif6, resembles the translation elongation factor eEF2, we suggest that assembly of the stalk recruits Efl1, triggering a step in 60S biogenesis that mimics aspects of translocation. Efl1 could thereby provide a mechanism to functionally check the nascent Subunit. Finally, the release of Tif6 is a prerequisite for the release of the nuclear export adaptor Nmd3. Establishing this pathway provides an important conceptual framework for understanding ribosome maturation.
Molecular Biology of the Cell, 2008Co-Authors: Nai Jung Hung, Samir S. Patel, Kara J. Helmke, Arlen W. JohnsonAbstract:
We previously showed that nuclear export of the large (60S) Ribosomal Subunit relies on Nmd3 in a Crm1-dependent manner. Recently the general mRNA export factor, the Mtr2/Mex67 heterodimer, was shown to act as an export receptor in parallel with Crm1. These observations raise the possibility that nuclear export of the 60S Subunit in Saccharomyces cerevisiae requires multiple export receptors. Here, we show that the previously characterized 60S Subunit biogenesis factor, Arx1, also acts as an export receptor for the 60S Subunit. We found that deletion of ARX1 was synthetic lethal with nmd3 and mtr2 mutants and was synthetic sick with several nucleoporin mutants. Deletion of ARX1 led to accumulation of pre-60S particles in the nucleus that were enriched for Nmd3, Crm1, Mex67, and Mtr2, suggesting that in the absence of Arx1, 60S export is impaired even though the Subunit is loaded with export receptors. Finally, Arx1 interacted with several nucleoporins in yeast two-hybrid as well as in vitro assays. These results show that Arx1 can directly bridge the interaction between the pre-60S particle and the NPC and thus is a third export receptor for the 60S Subunit in yeast.
Journal of Biological Chemistry, 2007Co-Authors: Matthew West, John Hedges, Arlen W. JohnsonAbstract:
Abstract Nuclear export of the large (60S) Ribosomal Subunit depends on the adapter protein Nmd3 to provide a nuclear export signal (NES). The leucine-rich NES is recognized by the export receptor Crm1 to mediate export via interaction with the nuclear pore complex (NPC). Here, we show that certain mutant Nmd3 proteins that are impaired for binding to the 60S Subunit accumulate at the nuclear envelope. These mutant proteins also show enhanced binding to Crm1, both in vivo and in vitro. Although their interaction with the NPC is dependent on recognition of the NES by Crm1, their interaction with Crm1 is not strictly dependent on RanGTP. Using a collection of GFP-tagged nucleoporin mutants, we identified several nucleoporins, including components of the Nup82 complex that copurified with the mutant Nmd3. The Nup82 complex is on the cytoplasmic face of the NPC and has previously been shown to be important as a terminal binding site for Crm1-mediated export. Mutations in the Nup82 complex led to accumulation of wild-type Nmd3 in the nucleoplasm, suggesting that the interaction of mutant Nmd3 with the Nup82 complex reflects a defect in the bona fide export pathway for the 60S Subunit. These results suggest that in the absence of the ribosome, Nmd3 is not efficiently released from Crm1 at the NPC.
Bernard L. Trumpower - One of the best experts on this subject based on the ideXlab platform.
RPL29 codes for a non-essential protein of the 60S Ribosomal Subunit in Saccharomyces cerevisiae and exhibits synthetic lethality with mutations in genes for proteins required for Subunit coupling.Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 2002Co-Authors: Marie Laure Delabre, Jacques J. Kessl, Spyridoula Karamanou, Bernard L. TrumpowerAbstract:
RPL29 (YFR032c-a) is a non-essential gene that codes for a 60S Ribosomal Subunit protein in Saccharomyces cerevisiae. Deletion of RPL29 leads to a moderate accumulation of half-mer polysomes with little or no change in the amounts of free 60S Subunits. In vitro translation and the growth rate are also delayed in the Δrpl29 strain. Such a phenotype is characteristic of mutants defective in 60S to 40S Subunit joining. The Δrpl29 strain exhibits synthetic lethality with mutations in RPL10, the gene encoding an essential 60S Ribosomal Subunit protein that is required for 60S to 40S Subunit joining. The Δrpl29 strain also exhibits synthetic lethality with RSA1, a gene encoding a nucleoplasmic protein required for the loading of Rpl10p onto the 60S Subunit . Over-expression of RPL10 suppresses the half-mer phenotype of the Δrpl29 strain, but does not correct the growth defect of the deletion strain. We conclude that absence of Rpl29p impairs proper assembly of proteins onto the 60S Subunit and that this retards Subunit joining and additionally retards protein synthesis subsequent to Subunit joining.
Heterologous complementation reveals that mutant alleles of QSR1 render 60S Ribosomal Subunits unstable and translationally inactiveNucleic Acids Research, 1998Co-Authors: Frederick A Dick, Bernard L. TrumpowerAbstract:
QSR1 is a highly conserved gene which encodes a 60S Ribosomal Subunit protein that is required for joining of large and small Ribosomal Subunits. In this report we demonstrate heterologous complementation of a yeast QSR1 deletion strain with both the human and corn homologs and show that the human and corn proteins are assembled into hybrid yeast/human and yeast/corn ribosomes. While the homologous genes complement lethality of the QSR1 deletion, they also result in a diminished growth rate. Analyses of the translation rates of ribosomes containing the human and corn proteins reveal a partial loss of function. Velocity gradient analyses of the hybrid ribosomes after exposure to high concentrations of salt indicate that the decreased activity is due to lability of the hybrid 60S Subunits.
Exchangeability of Qsr1p, a large Ribosomal Subunit protein required for Subunit joining, suggests a novel translational regulatory mechanismFEBS Letters, 1997Co-Authors: Frederick A Dick, Dominic P Eisinger, Bernard L. TrumpowerAbstract:
Qsr1p is a 60S Ribosomal Subunit protein that is necessary for joining of large and small Ribosomal Subunits and is also one of the last proteins assembled onto the 60S Ribosomal Subunit in the cytoplasm. The finding that Qsr1p is identical to L7, a protein previously shown to cycle on and off large Ribosomal Subunits in the cytoplasm, suggests that the addition of Qsr1p onto the 60S Ribosomal Subunit could be utilized as a translational regulatory mechanism by limiting the supply of functional 60S Subunits.
Molecular and Cellular Biology, 1997Co-Authors: Dominic P Eisinger, Frederick A Dick, Bernard L. TrumpowerAbstract:
QSR1 is a recently discovered, essential Saccharomyces cerevisiae gene, which encodes a 60S Ribosomal Subunit protein. Thirty-one unique temperature-sensitive alleles of QSR1 were generated by regional codon randomization within a conserved 20-amino-acid sequence of the QSR1-encoded protein. The temperature-sensitive mutants arrest as viable, large, unbudded cells 24 to 48 h after a shift to 37 degrees C. Polysome and Ribosomal Subunit analysis by velocity gradient centrifugation of lysates from temperature-sensitive qsr1 mutants and from cells in which Qsr1p was depleted by down regulation of an inducible promoter revealed the presence of half-mer polysomes and a large pool of free 60S Subunits that lack Qsr1p. In vitro Subunit-joining assays and analysis of a mutant conditional for the synthesis of Qsr1p demonstrate that 60S Subunits devoid of Qsr1p are unable to join with 40S Subunits whereas 60S Subunits that contain either wild-type or mutant forms of the protein are capable of Subunit joining. The defective 60S Subunits result from a reduced association of mutant Qsr1p with 60S Subunits. These results indicate that Qsr1p is required for Ribosomal Subunit joining.