scholarly journals e IF 5 B employs a novel domain release mechanism to catalyze ribosomal subunit joining

2014 ◽  
Vol 33 (10) ◽  
pp. 1177-1191 ◽  
Author(s):  
Bernhard Kuhle ◽  
Ralf Ficner
Keyword(s):  
Ribosomal Subunit ◽  
Release Mechanism ◽  
Subunit Joining

scholarly journals Characterization of the nuclear export adaptor protein Nmd3 in association with the 60S ribosomal subunit

2010 ◽  
Vol 189 (7) ◽  
pp. 1079-1086 ◽  
Author(s):  
Jayati Sengupta ◽  
Cyril Bussiere ◽  
Jesper Pallesen ◽  
Matthew West ◽  
Arlen W. Johnson ◽  
...  
Keyword(s):  
Binding Site ◽  
Nuclear Export ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Adaptor Protein ◽  
Structural Data ◽  
Release Mechanism ◽  
Nuclear Pore ◽  
Subunit Joining

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.

scholarly journals Coupled Release of Eukaryotic Translation Initiation Factors 5B and 1A from 80S Ribosomes following Subunit Joining

2007 ◽  
Vol 27 (6) ◽  
pp. 2384-2397 ◽  
Author(s):  
Jeanne M. Fringer ◽  
Michael G. Acker ◽  
Christie A. Fekete ◽  
Jon R. Lorsch ◽  
Thomas E. Dever
Keyword(s):  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
Cell Extracts ◽  
Eukaryotic Translation Initiation ◽  
Subunit Joining

ABSTRACT The translation initiation GTPase eukaryotic translation initiation factor 5B (eIF5B) binds to the factor eIF1A and catalyzes ribosomal subunit joining in vitro. We show that rapid depletion of eIF5B in Saccharomyces cerevisiae results in the accumulation of eIF1A and mRNA on 40S subunits in vivo, consistent with a defect in subunit joining. Substituting Ala for the last five residues in eIF1A (eIF1A-5A) impairs eIF5B binding to eIF1A in cell extracts and to 40S complexes in vivo. Consistently, overexpression of eIF5B suppresses the growth and translation initiation defects in yeast expressing eIF1A-5A, indicating that eIF1A helps recruit eIF5B to the 40S subunit prior to subunit joining. The GTPase-deficient eIF5B-T439A mutant accumulated on 80S complexes in vivo and was retained along with eIF1A on 80S complexes formed in vitro. Likewise, eIF5B and eIF1A remained associated with 80S complexes formed in the presence of nonhydrolyzable GDPNP, whereas these factors were released from the 80S complexes in assays containing GTP. We propose that eIF1A facilitates the binding of eIF5B to the 40S subunit to promote subunit joining. Following 80S complex formation, GTP hydrolysis by eIF5B enables the release of both eIF5B and eIF1A, and the ribosome enters the elongation phase of protein synthesis.

scholarly journals Mechanism of eIF6-mediated Inhibition of Ribosomal Subunit Joining

2010 ◽  
Vol 285 (20) ◽  
pp. 14848-14851 ◽  
Author(s):  
Marco Gartmann ◽  
Michael Blau ◽  
Jean-Paul Armache ◽  
Thorsten Mielke ◽  
Maya Topf ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Subunit Joining

Mechanism of ribosomal subunit joining during eukaryotic translation initiation

2008 ◽  
Vol 36 (4) ◽  
pp. 653-657 ◽  
Author(s):  
Michael G. Acker ◽  
Jon R. Lorsch
Keyword(s):  
Translation Initiation ◽  
Control Point ◽  
Ribosomal Subunit ◽  
Eukaryotic Translation ◽  
Translational Machinery ◽  
Biophysical Studies ◽  
Eukaryotic Translation Initiation ◽  
A Cell ◽  
Subunit Joining ◽  
Compare And Contrast

Decades of research have yielded significant insight into the mechanism by which a cell translates an mRNA into the encoded protein. However many of the molecular details of the process remain a mystery. Translation initiation is an important control point in gene expression, and misregulation can lead to diseases such as cancer. A better understanding of the mechanism of translation initiation is imperative for the development of novel therapeutic agents. Recently, a combination of genetic, biochemical and biophysical studies has begun to shed light on how, at a molecular level, the translational machinery initiates protein synthesis. In the present review, we briefly compare and contrast the initiation pathways utilized by bacteria, archaea and eukaryotes, and then focus on translation initiation in eukaryotes and recent advances in our understanding of the subunit joining step of the process.

scholarly journals Involvement of protein IF2 N domain in ribosomal subunit joining revealed from architecture and function of the full-length initiation factor

2013 ◽  
Vol 110 (39) ◽  
pp. 15656-15661 ◽  
Author(s):  
A. Simonetti ◽  
S. Marzi ◽  
I. M. L. Billas ◽  
A. Tsai ◽  
A. Fabbretti ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Full Length ◽  
Initiation Factor ◽  
And Function ◽  
Subunit Joining

SBDS and eIF6 Modulate Ribosome Subunit Joining in Shwachman Diamond Syndrome,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3438-3438
Author(s):  
Nicholas Burwick ◽  
Scott Coats ◽  
Akiko Shimamura
Keyword(s):  
Stromal Cells ◽  
Ribosome Biogenesis ◽  
Conflicts Of Interest ◽  
Ribosomal Subunit ◽  
Marrow Stromal Cells ◽  
Vitro Assay ◽  
Amino Terminal ◽  
Shwachman Diamond Syndrome ◽  
Subunit Joining

Abstract 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.

Lessons from the Mouse Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. SCI-35-SCI-35
Author(s):  
Johanna M. Rommens ◽  
Marina E. Tourlakis ◽  
Holly Liu ◽  
Rikesh Gandhi ◽  
Peter R. Durie
Keyword(s):  
Protein Synthesis ◽  
Conflicts Of Interest ◽  
Perinatal Death ◽  
Fetal Liver ◽  
Ribosomal Subunit ◽  
Growth Impairment ◽  
Protein Synthesis Machinery ◽  
P53 Activation ◽  
Subunit Joining ◽  
The Brain

Abstract Shwachman-Diamond syndrome (SDS) is a member of the growing list of inherited diseases that result from mutations in genes that encode components of the protein synthesis machinery. Collectively referred to as ribosomopathies, these diseases have selective organ involvement and serious hematological issues with increased susceptibility to malignancy. SDS is caused by mutations in the highly conserved SBDS gene. Recent studies have implicated SBDS in facilitating ribosomal subunit joining for translation initiation. We study mouse models with disease-associated alleles to determine the consequences of loss of Sbds and gain insight into ribosomopathy phenotypes. Mice that are homozygous for a disease-associated allele (SbdsR126T/R126T) die at birth. Growth issues and developmental delay are evident in mid-to-late-stage embryos, and analysis of ribosome profiles indicate disturbed protein synthesis in all tissues examined. SDS-related pathologies in the fetal liver, bone, and brain are also observed. For example, flow cytometry analyses and culturing of fetal liver cells show growth impairment of myeloid lineage progenitors. Ossification is delayed in the developing phalanges. Poor proliferation is evident in early neurons and death due to apoptosis occurs in differentiating neurons, as early as E14.5. As with the brain, the fetal spleen also shows disproportionate growth restriction. These responses reflect varying underlying molecular pathways that hinge on p53 activation. Breeding to Trp53 (p53) deficient mice attenuates the SDS phenotypes but does not rescue the overall growth impairment or the perinatal death. Loss of Sbds leads to a range of organ responses, with engagement of stress pathways at different times and stages. As the severity of the organ pathology does not correlate directly with alteration of 80S or polysome component peak levels in ribosome profiles, we conclude that the organ phenotype reflects limitations in gene expression and timing of expression, or results from disturbed regulation that is specific to cell type and respective protein synthesis demand. Disclosures: No relevant conflicts of interest to declare.

scholarly journals EFL1 mutations impair eIF6 release to cause Shwachman-Diamond syndrome

Blood ◽  
2019 ◽  
Vol 134 (3) ◽  
pp. 277-290 ◽  
Author(s):  
Shengjiang Tan ◽  
Laëtitia Kermasson ◽  
Angela Hoslin ◽  
Pekka Jaako ◽  
Alexandre Faille ◽  
...  
Keyword(s):  
Hematological Malignancies ◽  
Bone Marrow Failure ◽  
Ribosomal Subunit ◽  
Elongation Factor ◽  
Protein Translation ◽  
Allosteric Regulator ◽  
Shwachman Diamond Syndrome ◽  
Biallelic Mutations ◽  
Key Aspects ◽  
Subunit Joining

Abstract Shwachman-Diamond syndrome (SDS) is a recessive disorder typified by bone marrow failure and predisposition to hematological malignancies. SDS is predominantly caused by deficiency of the allosteric regulator Shwachman-Bodian-Diamond syndrome that cooperates with elongation factor-like GTPase 1 (EFL1) to catalyze release of the ribosome antiassociation factor eIF6 and activate translation. Here, we report biallelic mutations in EFL1 in 3 unrelated individuals with clinical features of SDS. Cellular defects in these individuals include impaired ribosomal subunit joining and attenuated global protein translation as a consequence of defective eIF6 eviction. In mice, Efl1 deficiency recapitulates key aspects of the SDS phenotype. By identifying biallelic EFL1 mutations in SDS, we define this leukemia predisposition disorder as a ribosomopathy that is caused by corruption of a fundamental, conserved mechanism, which licenses entry of the large ribosomal subunit into translation.

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