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Linda L. Spremulli - One of the best experts on this subject based on the ideXlab platform.
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Structure of Human Mitochondrial Translation Initiation Factor 3 Bound to the Small Ribosomal Subunit.
iScience, 2019Co-Authors: Ravi K. Koripella, Linda L. Spremulli, Manjuli R. Sharma, Emdadul Haque, Paul Risteff, Rajendra K. AgrawalAbstract:Summary The human mitochondrial translational Initiation Factor 3 (IF3mt) carries mitochondrial-specific amino acid extensions at both its N and C termini (N- and C-terminal extensions [NTE and CTE, respectively]), when compared with its eubacterial counterpart. Here we present 3.3- to 3.5-A-resolution cryoelectron microscopic structures of the mammalian 28S mitoribosomal subunit in complex with human IF3mt. Unique contacts observed between the 28S subunit and N-terminal domain of IF3mt explain its unusually high affinity for the 28S subunit, whereas the position of the mito-specific NTE suggests NTE's role in binding of initiator tRNA to the 28S subunit. The location of the C-terminal domain (CTD) clarifies its anti-association activity, whereas the orientation of the mito-specific CTE provides a mechanistic explanation for its role in destabilizing initiator tRNA in the absence of mRNA. Furthermore, our structure hints at a possible role of the CTD in recruiting leaderless mRNAs for translation Initiation. Our findings highlight unique features of IF3mt in mitochondrial translation Initiation.
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Fidelity of translation in the presence of mammalian mitochondrial Initiation Factor 3.
Mitochondrion, 2017Co-Authors: Shreya Ahana Ayyub, S L Aswathy, Divya Dobriyal, Srinivas Aluri, Linda L. Spremulli, Umesh VarshneyAbstract:Initiation Factor 3 (IF3) is a conserved translation Factor. Mutations in mitochondrial IF3 (IF3mt) have been implicated in disease pathology. Escherichia coli infCΔ55, compromised for IF3 activity, has provided an excellent heterologous system for IF3mt structure-function analysis. IF3mt allowed promiscuous Initiation from AUA, AUU and ACG codons but avoided Initiation with initiator tRNAs lacking the conserved 3GC pairs in their anticodon stems. Expression of IF3mt N-terminal domain, or IF3mt devoid of its typical N-, and C-terminal extensions improved fidelity of Initiation in E. coli. The observations suggest that the IF3mt terminal extensions relax the fidelity of translational Initiation in mitochondria.
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Contacts between mammalian mitochondrial translational Initiation Factor 3 and ribosomal proteins in the small subunit.
Biochimica et biophysica acta, 2011Co-Authors: Emdadul Haque, Hasan Koc, Huseyin Cimen, Emine C. Koc, Linda L. SpremulliAbstract:Mammalian mitochondrial translational Initiation Factor 3 (IF3mt) binds to the small subunit of the ribosome displacing the large subunit during the Initiation of protein biosynthesis. About half of the proteins in mitochondrial ribosomes have homologs in bacteria while the remainder are unique to the mitochondrion. To obtain information on the ribosomal proteins located near the IF3mt binding site, cross-linking studies were carried out followed by identification of the cross-linked proteins by mass spectrometry. IF3mt cross-links to mammalian mitochondrial homologs of the bacterial ribosomal proteins S5, S9, S10, and S18-2 and to unique mitochondrial ribosomal proteins MRPS29, MRPS32, MRPS36 and PTCD3 (Pet309) which has now been identified as a small subunit ribosomal protein. IF3mt has extensions on both the N- and C-termini compared to the bacterial Factors. Cross-linking of a truncated derivative lacking these extensions gives the same hits as the full length IF3mt except that no cross-links were observed to MRPS36. IF3 consists of two domains separated by a flexible linker. Cross-linking of the isolated N- and C-domains was observed to a range of ribosomal proteins particularly with the C-domain carrying the linker which showed significant cross-linking to several ribosomal proteins not found in prokaryotes.
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The interaction of mammalian mitochondrial translational Initiation Factor 3 with ribosomes: evolution of terminal extensions in IF3mt.
Nucleic acids research, 2007Co-Authors: Emdadul Haque, Domenick Grasso, Linda L. SpremulliAbstract:Mammalian mitochondrial Initiation Factor 3 (IF3mt) has a central region with homology to bacterial IF3. This homology region is preceded by an N-terminal extension and followed by a C-terminal extension. The role of these extensions on the binding of IF3mt to mitochondrial small ribosomal subunits (28S) was studied using derivatives in which the extensions had been deleted. The Kd for the binding of IF3mt to 28S subunits is ~30nM. Removal of either the N- or C-terminal extension has almost no effect on this value. IF3mt has very weak interactions with the large subunit of the mitochondrial ribosome (39S) (Kd=1.5kM). However, deletion of the extensions results in derivatives with significant affinity for 39S subunits (Kd=0.1220.25kM). IF3mt does not bind 55S monosomes, while the deletion derivative binds slightly to these particles. IF3mt is very effective in dissociating 55S ribosomes. Removal of the Nterminal extension has little effect on this activity. However, removal of the C-terminal extension leads to a complex dissociation pattern due to the high affinity of this derivative for 39S subunits. These data suggest that the extensions have evolved to ensure the proper dissociation of IF3mt from the 28S subunits upon 39S subunit joining.
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Overexpression and purification of mammalian mitochondrial translational Initiation Factor 2 and Initiation Factor 3.
Methods in enzymology, 2007Co-Authors: Domenick Grasso, Brooke E. Christian, Angela C. Spencer, Linda L. SpremulliAbstract:Abstract Two mammalian mitochondrial Initiation Factors have been identified. Initiation Factor 2 (IF2 mt ) selects the initiator tRNA (fMet-tRNA) and promotes its binding to the ribosome. Initiation Factor 3 (IF3 mt ) promotes the dissociation of the 55S mitochondrial ribosome into subunits and may play additional, less-well-understood, roles in Initiation complex formation. Native bovine IF2 mt was purified from liver a number of years ago. The yield of this Factor is very low making biochemical studies difficult. The cDNA for bovine IF2 mt was expressed in Escherichia coli under the control of the T7 polymerase promoter in a vector that provides a His 6 -tag at the C-terminus of the expressed protein. This Factor was expressed in E. coli and purified by chromatography on Ni-NTA resins. The expressed protein has a number of degradation products in partially purified preparations and this Factor is then further purified by high-performance liquid chromatography or gravity chromatography on anion exchange resins. IF3 mt has never been purified from any mammalian system. However, the cDNA for this protein can be identified in the expressed sequence tag (EST) libraries. The portion of the sequence encoding the region of human IF3 mt predicted to be present in the mitochondrially imported form of this Factor was cloned and expressed in E. coli using a vector that provides a C-terminal His 6 -tag. The tagged Factor is partially purified on Ni-NTA resins. However, a major proteolytic fragment arising from a defined cleavage of this protein is present in these preparations. This contaminant can be removed by a single step of high-performance liquid chromatography on a cation exchange resin. Alternatively, the mature form of IF3 mt can be purified by two sequential passes through a gravity S-Sepharose column.
John W. B. Hershey - One of the best experts on this subject based on the ideXlab platform.
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the p34cdc2 related cyclin dependent kinase 11 interacts with the p47 subunit of eukaryotic Initiation Factor 3 during apoptosis
Journal of Biological Chemistry, 2003Co-Authors: Jiaqi Shi, John W. B. Hershey, Yongmei Feng, Anne Christine Goulet, Richard R Vaillancourt, Nancy A Sachs, Mark A NelsonAbstract:Abstract Cyclin-dependent kinase 11 (CDK11; also named PITSLRE) is part of the large family of p34cdc2-related kinases whose functions appear to be linked with cell cycle progression, tumorigenesis, and apoptotic signaling. However, substrates of CDK11 during apoptosis have not been identified. We used a yeast two-hybrid screening strategy and identified eukaryotic Initiation Factor 3 p47 protein (eIF3 p47) as an interacting partner of caspase-processed C-terminal kinase domain of CDK11 (CDK11p46). We demonstrate that the eIF3 p47 can interact with CDK11 in vitro and in vivo, and the interaction can be strengthened by stimulation of apoptosis. EIF3 p47 contains a Mov34/JAB domain and appears to interact with CDK11p46 through this motif. We show in vitrothat the caspase-processed CDK11p46 can phosphorylate eIF3 p47 at a specific serine residue (Ser46) and that eIF3 p47 is phosphorylated in vivo during apoptosis. Purified recombinant CDK11p46 inhibited translation of a reporter gene in vitro in a dose-dependent manner. In contrast, a kinase-defective mutant CDK11p46M did not inhibit translation of the reporter gene. Stable expression of CDK11p46 in vivo inhibited the synthesis of a transfected luciferase reporter protein and overall cellular protein synthesis. These data provide insight into the cellular function of CDK11 during apoptosis.
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Malignant transformation by the eukaryotic translation Initiation Factor 3 subunit p48 (eIF3e)
FEBS letters, 2002Co-Authors: Greg L. Mayeur, John W. B. HersheyAbstract:Several components of translation, e.g. eIF4E and PKR, are implicated in cancer. The e-subunit (p48) of mammalian Initiation Factor 3 is encoded by the Int6 gene, a common site for integration of the mouse mammary tumor virus genome, leading to the production of a truncated eukaryotic Initiation Factor-3e (eIF3e). Stable expression of a truncated eIF3e in NIH 3T3 cells causes malignant transformation by four criteria: foci formation; anchorage independent growth; accelerated growth; and lack of contact inhibition. Stable expression of full-length eIF3e does not cause transformation. The truncated eIF3e also inhibits the onset of apoptosis caused by serum starvation.
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Characterization of the p33 Subunit of Eukaryotic Translation Initiation Factor-3 from Saccharomyces cerevisiae
The Journal of biological chemistry, 1999Co-Authors: Parisa Hanachi, John W. B. Hershey, Hans-peter VornlocherAbstract:Abstract Eukaryotic translation Initiation Factor-3 (eIF3) is a large multisubunit complex that binds to the 40 S ribosomal subunit and promotes the binding of methionyl-tRNAiand mRNA. The molecular mechanism by which eIF3 exerts these functions is incompletely understood. We report here the cloning and characterization of TIF35, the Saccharomyces cerevisiae gene encoding the p33 subunit of eIF3. p33 is an essential protein of 30,501 Da that is required in vivo for Initiation of protein synthesis. Glucose repression ofTIF35 expressed from a GAL1 promoter results in depletion of both the p33 and p39 subunits. Expression of histidine-tagged p33 in yeast in combination with Ni2+affinity chromatography allows the isolation of a complex containing the p135, p110, p90, p39, and p33 subunits of eIF3. The p33 subunit binds both mRNA and rRNA fragments due to an RNA recognition motif near its C terminus. Deletion of the C-terminal 71 amino acid residues causes loss of RNA binding, but expression of the truncated form as the sole source of p33 nevertheless supports the slow growth of yeast. These results indicate that the p33 subunit of eIF3 plays an important role in the Initiation phase of protein synthesis and that its RNA-binding domain is required for optimal activity.
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structure of cdnas encoding human eukaryotic Initiation Factor 3 subunits possible roles in rna binding and macromolecular assembly
Journal of Biological Chemistry, 1997Co-Authors: Katsura Asano, Hans-peter Vornlocher, William C. Merrick, Alan G. Hinnebusch, Nancy J Richtercook, John W. B. HersheyAbstract:Abstract The mammalian translation Initiation Factor 3 (eIF3), is a multiprotein complex of ∼600 kDa that binds to the 40 S ribosome and promotes the binding of methionyl-tRNAi and mRNA. cDNAs encoding 5 of the 10 subunits, namely eIF3-p170, -p116, -p110, -p48, and -p36, have been isolated previously. Here we report the cloning and characterization of human cDNAs encoding the major RNA binding subunit, eIF3-p66, and two additional subunits, eIF3-p47 and eIF3-p40. Each of these proteins is present in immunoprecipitates formed with affinity-purified anti-eIF3-p170 antibodies. Human eIF3-p66 shares 64% sequence identity with a hypothetical Caenorhabditis elegans protein, presumably the p66 homolog. Deletion analyses of recombinant derivatives of eIF3-p66 show that the RNA-binding domain lies within an N-terminal 71-amino acid region rich in lysine and arginine. The N-terminal regions of human eIF3-p40 and eIF3-p47 are related to each other and to 17 other eukaryotic proteins, including murine Mov-34, a subunit of the 26 S proteasome. Phylogenetic analyses of the 19 related protein sequences, called the Mov-34 family, distinguish five major subgroups, where eIF3-p40, eIF3-p47, and Mov-34 are each found in a different subgroup. The subunit composition of eIF3 appears to be highly conserved inDrosophila melanogaster, C. elegans, andArabidopsis thaliana, whereas only 5 homologs of the 10 subunits of mammalian eIF3 are encoded in S. cerevisiae.
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Structure of cDNAs Encoding Human Eukaryotic Initiation Factor 3 Subunits
1997Co-Authors: Katsura Asano, Hans-peter Vornlocher, Nancy J. Richter-cook, William C. Merrick, Alan G. Hinnebusch, John W. B. HersheyAbstract:The mammalian translation Initiation Factor 3 (eIF3), is a multiprotein complex of ;600 kDa that binds to the 40 S ribosome and promotes the binding of methionyltRNA i and mRNA. cDNAs encoding 5 of the 10 subunits, namely eIF3-p170, -p116, -p110, -p48, and -p36, have been isolated previously. Here we report the cloning and characterization of human cDNAs encoding the major RNA binding subunit, eIF3-p66, and two additional subunits, eIF3-p47 and eIF3-p40. Each of these proteins is present in immunoprecipitates formed with affinity-purified anti-eIF3-p170 antibodies. Human eIF3-p66 shares 64% sequence identity with a hypothetical Caenorhabditis elegans protein, presumably the p66 homolog. Deletion analyses of recombinant derivatives of eIF3-p66 show that the RNA-binding domain lies within an Nterminal 71-amino acid region rich in lysine and arginine. The N-terminal regions of human eIF3-p40 and eIF3-p47 are related to each other and to 17 other eukaryotic proteins, including murine Mov-34, a subunit of the 26 S proteasome. Phylogenetic analyses of the 19 related protein sequences, called the Mov-34 family, distinguish five major subgroups, where eIF3-p40, eIF3p47, and Mov-34 are each found in a different subgroup. The subunit composition of eIF3 appears to be highly conserved in Drosophila melanogaster, C. elegans, and Arabidopsis thaliana, whereas only 5 homologs of the 10 subunits of mammalian eIF3 are encoded in S. cerevisiae.
Piotr Kamenski - One of the best experts on this subject based on the ideXlab platform.
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Yeast Mitochondrial Translation Initiation Factor 3 Interacts with Pet111p to Promote COX2 mRNA Translation.
International journal of molecular sciences, 2020Co-Authors: I. V. Chicherin, S. A. Levitskii, Maria V. Baleva, Igor A. Krasheninnikov, M. V. Patrushev, Piotr KamenskiAbstract:Mitochondrial genomes code for several core components of respiratory chain complexes. Thus, mitochondrial translation is of great importance for the organelle as well as for the whole cell. In yeast, mitochondrial translation Initiation Factor 3, Aim23p, is not essential for the organellar protein synthesis; however, its absence leads to a significant quantitative imbalance of the mitochondrial translation products. This fact points to a possible specific action of Aim23p on the biosynthesis of some mitochondrial protein species. In this work, we examined such peculiar effects of Aim23p in relation to yeast mitochondrial COX2 mRNA translation. We show that Aim23p is indispensable to this process. According to our data, this is mediated by Aimp23p interaction with the known specific Factor of the COX2 mRNA translation, Pet111p. If there is no Aim23p in the yeast cells, an increased amount of Pet111p ensures proper COX2 mRNA translation. Our results demonstrate the additional non-canonical function of Initiation Factor 3 in yeast mitochondrial translation.
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Biological and Evolutionary Significance of Terminal Extensions of Mitochondrial Translation Initiation Factor 3
International journal of molecular sciences, 2018Co-Authors: Ksenia Derbikova, I. V. Chicherin, S. A. Levitskii, Maria V. Baleva, Igor A. Krasheninnikov, Anton Kuzmenko, Maria Klimontova, Piotr KamenskiAbstract:Protein biosynthesis in mitochondria is organized in a bacterial manner. However, during evolution, mitochondrial translation mechanisms underwent many organelle-specific changes. In particular, almost all mitochondrial translation Factors, being orthologous to bacterial proteins, are characterized by some unique elements of primary or secondary structure. In the case of the organellar Initiation Factor 3 (IF3), these elements are several dozen amino acids long N- and C-terminal extensions. This study focused on the terminal extensions of baker’s yeast mitochondrial IF3, Aim23p. By in vivo deletion and complementation analysis, we show that at least one extension is necessary for Aim23p function. At the same time, human mitochondrial IF3 is fully functional in yeast mitochondria even without both terminal extensions. While Escherichia coli IF3 itself is poorly active in yeast mitochondria, adding Aim23p terminal extensions makes the resulting chimeric protein as functional as the cognate Factor. Our results show that the terminal extensions of IF3 have evolved as the “adaptors” that accommodate the translation Factor of bacterial origin to the evolutionary changed protein biosynthesis system in mitochondria.
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60S dynamic state of bacterial ribosome is fixed by yeast mitochondrial Initiation Factor 3.
PeerJ, 2018Co-Authors: S. A. Levitskii, I. V. Chicherin, Maria V. Baleva, Igor A. Krasheninnikov, Ksenia Derbikova, Anton Kuzmenko, Andrey V. Golovin, Piotr KamenskiAbstract:The processes of association and dissociation of ribosomal subunits are of great importance for the protein biosynthesis. The mechanistic details of these processes, however, are not well known. In bacteria, upon translation termination, the ribosome dissociates into subunits which is necessary for its further involvement into new Initiation step. The dissociated state of the ribosome is maintained by Initiation Factor 3 (IF3) which binds to free small subunits and prevents their premature association with large subunits. In this work, we have exchanged IF3 in Escherichia coli cells by its ortholog from Saccharomyces cerevisiae mitochondria (Aim23p) and showed that yeast protein cannot functionally substitute the bacterial one and is even slightly toxic for bacterial cells. Our in vitro experiments have demonstrated that Aim23p does not split E. coli ribosomes into subunits. Instead, it fixes a state of ribosomes characterized by sedimentation coefficient about 60S which is not a stable structure but rather reflects a shift of dynamic equilibrium between associated and dissociated states of the ribosome. Mitochondria-specific terminal extensions of Aim23p are necessary for "60S state" formation, and molecular modeling results point out that these extensions might stabilize the position of the protein on the bacterial ribosome.
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An intermediate state of bacterial ribosome dissociation is fixed by yeast mitochondrial Initiation Factor 3
2018Co-Authors: S. A. Levitskii, I. V. Chicherin, Maria V. Baleva, Igor A. Krasheninnikov, Ksenia Derbikova, Anton Kuzmenko, Andrey V. Golovin, Piotr KamenskiAbstract:The processes of association and dissociation of ribosomal subunits are of great importance for the protein biosynthesis. The mechanistic details of these processes, however, are not well known. In bacteria, upon translation termination, ribosome dissociates into subunits which is necessary for its further involvement into new Initiation step. The dissociated state of ribosome is maintained by Initiation Factor 3 (IF3) which binds to free small subunits and prevents their premature association with the large subunits. In this work, we have exchanged IF3 in E.coli cells by its ortholog from Saccharomyces cerevisiae mitochondria (Aim23p) and showed that yeast protein cannot functionally substitute the bacterial one and is even slightly toxic for bacterial cells. Our in vitro experiments have demonstrated that Aim23p does not split E.coli ribosomes into subunits. Instead, it fixes an intermediate state of ribosomes dissociation characterized by sedimentation coefficient about 60S. Using molecular modeling, we show that such fixation is due to mitochondria-specific terminal extensions of Aim23p that stabilize the position of the protein on the bacterial ribosome.
Honggang Cheng - One of the best experts on this subject based on the ideXlab platform.
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Eukaryotic translation Initiation Factor 3 subunit G promotes human colorectal cancer.
American journal of translational research, 2019Co-Authors: Chenggang Yang, Yanbo Zhang, Honggang ChengAbstract:In this study, we investigated the role of eukaryotic translation Initiation Factor 3 subunit G (EIF3G) in colorectal cancer. Immunohistochemical analysis showed higher EIF3G expression in stage IV human colorectal cancer tissues than in adjacent normal tissues (P
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Eukaryotic translation Initiation Factor 3 subunit G promotes human colorectal cancer
Oncotarget, 2018Co-Authors: Chenggang Yang, Yanbo Zhang, Honggang ChengAbstract:// Chenggang Yang 1 , Yanbo Zhang 1 , Wenfeng Du 1 , Honggang Cheng 1 and Chaobin Li 1 1 Department of Gastrointestinal Surgery, Liaocheng People’s Hospital, Liaocheng, China Correspondence to: Chenggang Yang, email: bakerham123@163.com Keywords: EIF3G; colorectal cancer; proliferation; apoptosis; autophagy Received: April 18, 2017 Accepted: November 12, 2017 Published: January 02, 2018 ABSTRACT In this study, we investigated the role of eukaryotic translation Initiation Factor 3 subunit G (EIF3G) in colorectal cancer. Immunohistochemical analysis showed higher EIF3G expression in stage IV human colorectal cancer tissues than in adjacent normal tissues ( P < 0.01). EIF3G shRNA knockdown in HCT116 colon cancer cells reduced proliferation and increased apoptosis as compared to control. EIF3G knockdown also increased autophagy and reduced mTOR signaling, as evidenced by low phospho-AKT, phospho-S6K and phospho-4EBP1 levels. Functional experiments indicated that overexpression of EIF3G promoted HCT-116 cells proliferation, migration and xenograft tumor growth. Finally, we observed lower xenograft tumor weights and volumes with EIF3G-silenced HCT116 cells than with control cells. These findings demonstrate that EIF3G promotes colon cancer growth and is a potential therapeutic target.
Chenggang Yang - One of the best experts on this subject based on the ideXlab platform.
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Eukaryotic translation Initiation Factor 3 subunit G promotes human colorectal cancer.
American journal of translational research, 2019Co-Authors: Chenggang Yang, Yanbo Zhang, Honggang ChengAbstract:In this study, we investigated the role of eukaryotic translation Initiation Factor 3 subunit G (EIF3G) in colorectal cancer. Immunohistochemical analysis showed higher EIF3G expression in stage IV human colorectal cancer tissues than in adjacent normal tissues (P
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Eukaryotic translation Initiation Factor 3 subunit G promotes human colorectal cancer
Oncotarget, 2018Co-Authors: Chenggang Yang, Yanbo Zhang, Honggang ChengAbstract:// Chenggang Yang 1 , Yanbo Zhang 1 , Wenfeng Du 1 , Honggang Cheng 1 and Chaobin Li 1 1 Department of Gastrointestinal Surgery, Liaocheng People’s Hospital, Liaocheng, China Correspondence to: Chenggang Yang, email: bakerham123@163.com Keywords: EIF3G; colorectal cancer; proliferation; apoptosis; autophagy Received: April 18, 2017 Accepted: November 12, 2017 Published: January 02, 2018 ABSTRACT In this study, we investigated the role of eukaryotic translation Initiation Factor 3 subunit G (EIF3G) in colorectal cancer. Immunohistochemical analysis showed higher EIF3G expression in stage IV human colorectal cancer tissues than in adjacent normal tissues ( P < 0.01). EIF3G shRNA knockdown in HCT116 colon cancer cells reduced proliferation and increased apoptosis as compared to control. EIF3G knockdown also increased autophagy and reduced mTOR signaling, as evidenced by low phospho-AKT, phospho-S6K and phospho-4EBP1 levels. Functional experiments indicated that overexpression of EIF3G promoted HCT-116 cells proliferation, migration and xenograft tumor growth. Finally, we observed lower xenograft tumor weights and volumes with EIF3G-silenced HCT116 cells than with control cells. These findings demonstrate that EIF3G promotes colon cancer growth and is a potential therapeutic target.