scholarly journals DDX3X and DDX3Y are redundant in protein synthesis

2020 ◽  
Author(s):  
Srivats Venkataramanan ◽  
Lorenzo Calviello ◽  
Kevin Wilkins ◽  
Stephen N. Floor
Keyword(s):  
Protein Synthesis ◽  
Developmental Disorders ◽  
Large Fraction ◽  
Ribosome Profiling ◽  
Variable Expression ◽  
Dead Box ◽  
Dependent Protein ◽  
The Central Nervous System ◽  
Male Specific

AbstractDDX3 is a DEAD-box RNA helicase that regulates translation and is encoded by the X- and Y-linked paralogs DDX3X and DDX3Y. While DDX3X is ubiquitously expressed in human tissues and essential for viability, DDX3Y is male-specific and shows lower and more variable expression than DDX3X in somatic tissues. Heterozygous genetic lesions in DDX3X mediate a class of developmental disorders called DDX3X syndrome, while loss of DDX3Y is implicated in male infertility. One possible explanation for female-bias in DDX3X syndrome is that DDX3Y encodes a polypeptide with different biochemical activity. In this study, we use ribosome profiling and in vitro translation to demonstrate that the X- and Y-linked paralogs of DDX3 play functionally redundant roles in translation. We find that transcripts that are sensitive to DDX3X depletion or mutation are rescued by complementation with DDX3Y. Our data indicate that DDX3X and DDX3Y proteins can functionally complement each other in human cells. DDX3Y is not expressed in a large fraction of the central nervous system. These findings suggest that expression differences, not differences in paralog-dependent protein synthesis, underlie the sex-bias of DDX3X-associated diseases.

scholarly journals DDX3X and DDX3Y are redundant in protein synthesis

RNA ◽  
2021 ◽  
pp. rna.078926.121
Author(s):  
Srivats Venkataramanan ◽  
Margaret Gadek ◽  
Lorenzo Calviello ◽  
Kevin Wilkins ◽  
Stephen Floor
Keyword(s):  
Protein Synthesis ◽  
Developmental Disorders ◽  
Large Fraction ◽  
Mrna Translation ◽  
Ribosome Profiling ◽  
In Vitro Translation ◽  
Variable Expression ◽  
Dependent Protein ◽  
Male Specific

DDX3 is a DEAD-box RNA helicase that regulates translation and is encoded by the X- and Y-linked paralogs DDX3X and DDX3Y. While DDX3X is ubiquitously expressed in human tissues and essential for viability, DDX3Y is male-specific and shows lower and more variable expression than DDX3X in somatic tissues. Heterozygous genetic lesions in DDX3X mediate a class of developmental disorders called DDX3X syndrome, while loss of DDX3Y is implicated in male infertility. One possible explanation for female-bias in DDX3X syndrome is that DDX3Y encodes a polypeptide with different biochemical activity. In this study, we use ribosome profiling and in vitro translation to demonstrate that the X- and Y-linked paralogs of DDX3 play functionally redundant roles in translation. We find that transcripts that are sensitive to DDX3X depletion or mutation are rescued by complementation with DDX3Y. Our data indicate that DDX3X and DDX3Y proteins can functionally complement each other in the context of mRNA translation in human cells. DDX3Y is not expressed in a large fraction of the central nervous system. These findings suggest that expression differences, not differences in paralog-dependent protein synthesis, underlie the sex-bias of DDX3X-associated diseases.

scholarly journals Differences in size, structure and function of free and membrane-bound polyribosomes of rat liver. Evidence for a single class of membrane-bound polyribosomes

1977 ◽  
Vol 168 (1) ◽  
pp. 1-8 ◽  
Author(s):  
J C Ramsey ◽  
W J Steele
Keyword(s):  
Protein Synthesis ◽  
Rat Liver ◽  
Size Structure ◽  
Large Fraction ◽  
Liver Homogenate ◽  
Structure And Function ◽  
Structural And Functional Properties ◽  
Membrane Bound ◽  
And Function

Free loosely bound and tightly bound polyribosomes were separated from rat liver homogenate by salt extraction followed by differential centrifugation, and several of their structural and functional properties were compared to resolve the existence of loosely bound polyribosomes and verify the specificity of the separation. The free and loosely bound polyribosomes have similar sedimentation profiles and polyribosome contents, their subunit proteins have similar electrophoretic patterns and their products of protein synthesis in vitro show a close correspondence in size and amounts synthesized. In contrast, the tightly bound polyribosomes have different properties from those of the free and loosely bound polyribosomes; their average size is significantly smaller; their polyribosome content is higher; their 60 S-subunit proteins lack two components and contain four or more components not found elsewhere; their products of protein synthesis in vitro differ in size and amounts synthesized. These observations show that rat liver membranes entrap a large fraction of the free polyribosomes at low salt concentrations and that these polyribosomes are similar to those of the free-polyribosome fraction and are different from those of the tightly bound polyribosome fraction in size, structure and function.

scholarly journals Mechanism of action of purpuromycin

1992 ◽  
Vol 284 (1) ◽  
pp. 47-52 ◽  
Author(s):  
P Landini ◽  
E Corti ◽  
B P Goldstein ◽  
M Denaro
Keyword(s):  
Protein Synthesis ◽  
Rna Synthesis ◽  
Intact Cell ◽  
Acid Synthesis ◽  
Free System ◽  
Dna And Rna ◽  
Dna And Rna Synthesis ◽  
Dependent Protein ◽  
Elongation Step

Purpuromycin, an antibiotic active against both fungi and bacteria, shows different modes of action against these two kinds of micro-organisms; in Candida albicans it inhibits RNA synthesis, whereas in Bacillus subtilis protein synthesis is primarily affected, with DNA and RNA synthesis blocked at higher concentrations of the drug. In bacterial cell-free protein-synthesis systems, purpuromycin did not inhibit synthesis from endogenous mRNA (elongation of peptides initiated within the intact cell) but inhibited MS2-phase RNA-dependent protein synthesis (which requires initiation) by 50% at 0.1 mg/l. Poly(U)-directed polyphenylalanine synthesis was 50% inhibited by 20 mg of purpuromycin/l when added to a complete system; however, when purpuromycin was preincubated with ribosomes dissociated into 30 S and 50 S subunits, the concentration for 50% inhibition fell to 0.1 mg/l. By contrast, in a C. albicans cell-free system poly(U)-directed polyphenylalanine synthesis was partially inhibited only at 200 mg/l. Purpuromycin also inhibited polynucleotide synthesis in vitro in reactions using Escherichia coli or wheat-germ RNA polymerases or E. coli DNA polymerase I. We suggest that in bacteria the primary target of purpuromycin is on ribosomes and that its action precedes the elongation step of protein synthesis. The effect on nucleic acid synthesis in both fungi and bacteria may be due to interaction of purpuromycin with DNA.

scholarly journals Inhibition of Double-Stranded RNA-Dependent Protein Kinase PKR by Vaccinia Virus E3: Role of Complex Formation and the E3 N-Terminal Domain

1998 ◽  
Vol 18 (12) ◽  
pp. 7304-7316 ◽  
Author(s):  
Patrick R. Romano ◽  
Fan Zhang ◽  
Seng-Lai Tan ◽  
Minerva T. Garcia-Barrio ◽  
Michael G. Katze ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Kinase Domain ◽  
Yeast Two Hybrid ◽  
Dependent Protein Kinase ◽  
Double Stranded Rna ◽  
Dsrna Binding ◽  
Dependent Protein ◽  
Two Hybrid

ABSTRACT The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits protein synthesis by phosphorylating translation initiation factor 2α (eIF2α). Vaccinia virus E3Lencodes a dsRNA binding protein that inhibits PKR in virus-infected cells, presumably by sequestering dsRNA activators. Expression of PKR in Saccharomyces cerevisiae inhibits protein synthesis by phosphorylation of eIF2α, dependent on its two dsRNA binding motifs (DRBMs). We found that expression of E3 in yeast overcomes the lethal effect of PKR in a manner requiring key residues (Lys-167 and Arg-168) needed for dsRNA binding by E3 in vitro. Unexpectedly, the N-terminal half of E3, and residue Trp-66 in particular, also is required for anti-PKR function. Because the E3 N-terminal region does not contribute to dsRNA binding in vitro, it appears that sequestering dsRNA is not the sole function of E3 needed for inhibition of PKR. This conclusion was supported by the fact that E3 activity was antagonized, not augmented, by overexpressing the catalytically defective PKR-K296R protein containing functional DRBMs. Coimmunoprecipitation experiments showed that a majority of PKR in yeast extracts was in a complex with E3, whose formation was completely dependent on the dsRNA binding activity of E3 and enhanced by the N-terminal half of E3. In yeast two-hybrid assays and in vitro protein binding experiments, segments of E3 and PKR containing their respective DRBMs interacted in a manner requiring E3 residues Lys-167 and Arg-168. We also detected interactions between PKR and the N-terminal half of E3 in the yeast two-hybrid and λ repressor dimerization assays. In the latter case, the N-terminal half of E3 interacted with the kinase domain of PKR, dependent on E3 residue Trp-66. We propose that effective inhibition of PKR in yeast requires formation of an E3-PKR-dsRNA complex, in which the N-terminal half of E3 physically interacts with the protein kinase domain of PKR.

scholarly journals The Molecular Pharmacology of Pateamine A

2021 ◽  
Author(s):  
◽  
James Henry Matthews
Keyword(s):  
Protein Synthesis ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Mode Of Action ◽  
Protein Synthesis Inhibitors ◽  
Primary Target ◽  
Dead Box ◽  
Mammalian Cell Lines ◽  
Pateamine A

<p>Pateamine A is a cytotoxic terpenoid isolated from the marine sponge Mycale hentscheli that induces apoptosis in mammalian cell lines and is growth inhibitory to yeasts and fungi, yet shows no inhibitory action in prokaryotes. The targets of pateamine in mammalian cell lines were isolated and identified using a combination of affinity chromatography and mass spectrometry, putative targets included the DEAD-Box helicase eIF4A family of proteins, β-tubulin and actin. In vitro assessment of tubulin and actin polymerization showed pateamine was able to affect them only at high micromolar concentrations, whereas the effect on eIF4A in vitro was shown by others to occur at nanomolar concentrations. Additionally, pateamine was shown to inhibit cap-dependent protein synthesis in vivo, suggesting eIF4A as a primary target. The generation of a pateamine resistance-conferring mutation in the yeast eIF4A encoding gene TIF1, suggested further that eIF4A is a primary target in both mammalian and yeast cells, and allows the speculation of the position of the binding site for pateamine on the N-terminal lobe of eIF4A and the proposal of potential covalent interaction between this drug and its target. Given the size of the DEAD-Box helicase family, all of which share considerable homology with the eIF4As, FAL1 especially which is essential for rRNA maturation, a chemogenomic screen was performed in an attempt to establish the breadth of functional interactions of pateamine. The results of hierarchical clustering of these screen results suggest that pateamine has a mode-of-action distinct from other compounds screened previously, despite its effect on protein synthesis it failed to cluster with any other protein synthesis inhibitors regardless of their separate mechanisms, though, as a class, protein synthesis inhibitors were not found to form a discrete cluster in any of the variations of cluster analysis performed. Functional analysis, by GO term enrichment, of the genes whose deletions are hypersensitive to pateamine indicates that deletions of genes involved in numerous aspects of RNA metabolism affect pateamine sensitivity, however clear results regarding the involvement of FAL1 or any other non-eIF4A target in pateamine’s mode-of-action were not found.</p>

scholarly journals cAMP induces co-translational modification of proteins in IPC-81 cells

1999 ◽  
Vol 342 (2) ◽  
pp. 369-377 ◽  
Author(s):  
Randi HOVLAND ◽  
Anne P. DØSKELAND ◽  
Thor S. EIKHOM ◽  
Bernard ROBAYE ◽  
Stein O. DØSKELAND
Keyword(s):  
Protein Synthesis ◽  
Dependent Manner ◽  
Dependent Protein Kinase ◽  
Intact Cells ◽  
Leukaemia Cell Line ◽  
Camp Analogue ◽  
Dependent Protein ◽  
Induced Apoptosis

An elevated cAMP concentration results in growth arrest and protein synthesis-dependent apoptosis in the promyelocytic leukaemia cell line IPC-81. A comparison of two-dimensional gels of extracts from these cells labelled with [35S]methionine revealed that five distinct protein spots were induced by cAMP in a protein-synthesis-dependent manner. The spots seemed to result from the acidic shift of a precursor protein. The most abundant spot was phospho-actin. The spots induced by cAMP in intact cells were induced by cAMP-dependent protein kinase (cAPK) during the translation in vitro of mRNA from the leukaemia cells. The effect of cAPK was strictly co-translational, none of the spots being induced when cAPK was added after translation. This suggested that the protein spots arose by co-translational phosphorylation catalysed by cAPK. Two of the protein spots, phospho-actin and a protein with a molecular mass of 30 kDa and an isoelectric point of 4.5, were studied further with respect to expression. They were produced during the whole pre-apoptotic period, had cellular half-lives of several hours and were induced by the same concentrations of cAMP analogue that induced apoptosis. It is suggested that the accumulation of co-translationally modified proteins could be important for long-term cAMP signalling.

scholarly journals Evidence for misreading during tRNA-dependent protein synthesis in vitro.

1978 ◽  
Vol 253 (10) ◽  
pp. 3482-3486
Author(s):  
W.M. Holmes ◽  
G.W. Hatfield ◽  
E. Goldman
Keyword(s):  
Protein Synthesis ◽  
Dependent Protein

scholarly journals Abolition of stress-induced protein synthesis sensitizes leukemia cells to anthracycline-induced death

Blood ◽  
2008 ◽  
Vol 111 (5) ◽  
pp. 2866-2877 ◽  
Author(s):  
Gro Gausdal ◽  
Bjørn Tore Gjertsen ◽  
Emmet McCormack ◽  
Petra Van Damme ◽  
Randi Hovland ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Myelogenous Leukemia ◽  
Gene Products ◽  
Acute Myelogenous ◽  
Dependent Protein ◽  
Specific Mrnas ◽  
Optimal Effect ◽  
Proapoptotic Gene ◽  
Independent Translation

Anthracycline action has been thought to involve the neosynthesis of proapoptotic gene products and to therefore depend on protein synthesis for optimal effect. We found that inhibition of general, but not rapamycin-sensitive (cap-dependent), protein synthesis in the preapoptotic period enhanced anthracycline-induced acute myelogenous leukemia (AML) cell death, both in vitro and in several animal AML models. Pre-apoptotic anthracycline-exposed AML cells had altered translational specificity, with enhanced synthesis of a subset of proteins, including endoplasmatic reticulum chaperones. The altered translational specificity could be explained by perturbation (protein degradation, truncation, or dephosphorylation) of the cap-dependent translation initiation machinery and of proteins control-ing translation of specific mRNAs. We propose that judiciously timed inhibition of cap-independent translation is considered for combination therapy with anthracyclines in AML.

scholarly journals The Molecular Pharmacology of Pateamine A

2021 ◽  
Author(s):  
◽  
James Henry Matthews
Keyword(s):  
Protein Synthesis ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Mode Of Action ◽  
Protein Synthesis Inhibitors ◽  
Primary Target ◽  
Dead Box ◽  
Mammalian Cell Lines ◽  
Pateamine A

<p>Pateamine A is a cytotoxic terpenoid isolated from the marine sponge Mycale hentscheli that induces apoptosis in mammalian cell lines and is growth inhibitory to yeasts and fungi, yet shows no inhibitory action in prokaryotes. The targets of pateamine in mammalian cell lines were isolated and identified using a combination of affinity chromatography and mass spectrometry, putative targets included the DEAD-Box helicase eIF4A family of proteins, β-tubulin and actin. In vitro assessment of tubulin and actin polymerization showed pateamine was able to affect them only at high micromolar concentrations, whereas the effect on eIF4A in vitro was shown by others to occur at nanomolar concentrations. Additionally, pateamine was shown to inhibit cap-dependent protein synthesis in vivo, suggesting eIF4A as a primary target. The generation of a pateamine resistance-conferring mutation in the yeast eIF4A encoding gene TIF1, suggested further that eIF4A is a primary target in both mammalian and yeast cells, and allows the speculation of the position of the binding site for pateamine on the N-terminal lobe of eIF4A and the proposal of potential covalent interaction between this drug and its target. Given the size of the DEAD-Box helicase family, all of which share considerable homology with the eIF4As, FAL1 especially which is essential for rRNA maturation, a chemogenomic screen was performed in an attempt to establish the breadth of functional interactions of pateamine. The results of hierarchical clustering of these screen results suggest that pateamine has a mode-of-action distinct from other compounds screened previously, despite its effect on protein synthesis it failed to cluster with any other protein synthesis inhibitors regardless of their separate mechanisms, though, as a class, protein synthesis inhibitors were not found to form a discrete cluster in any of the variations of cluster analysis performed. Functional analysis, by GO term enrichment, of the genes whose deletions are hypersensitive to pateamine indicates that deletions of genes involved in numerous aspects of RNA metabolism affect pateamine sensitivity, however clear results regarding the involvement of FAL1 or any other non-eIF4A target in pateamine’s mode-of-action were not found.</p>

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