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PCI proteins eIF3e and eIF3m define distinct translation initiation factor 3 complexes.

Zhou C, Arslan F, Wee S, Krishnan S, Ivanov AR, Oliva A, Leatherwood J, Wolf DA - BMC Biol. (2005)

Bottom Line: Whereas neither eIF3e nor eIF3m contribute to the non-essential activities of CSN in cullin-RING ubiquitin ligase control, eif3m, unlike eif3e, is an essential gene required for global cellular protein synthesis and polysome formation.Using a ribonomic approach, this phenotypic distinction was correlated with a different set of mRNAs associated with the eIF3e and eIF3m complexes.We propose that the PCI proteins eIF3e and eIF3m define distinct eIF3 complexes that may assist in the translation of different sets of mRNAs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics and Complex Diseases, Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02115, USA. czhou@rics.bwh.harvard.edu

ABSTRACT

Background: PCI/MPN domain protein complexes comprise the 19S proteasome lid, the COP9 signalosome (CSN), and eukaryotic translation initiation factor 3 (eIF3). The eIF3 complex is thought to be composed of essential core subunits required for global protein synthesis and non-essential subunits that may modulate mRNA specificity. Interactions of unclear significance were reported between eIF3 subunits and PCI proteins contained in the CSN.

Results: Here, we report the unexpected finding that fission yeast has two distinct eIF3 complexes sharing common core subunits, but distinguished by the PCI proteins eIF3e and the novel eIF3m, which was previously annotated as a putative CSN subunit. Whereas neither eIF3e nor eIF3m contribute to the non-essential activities of CSN in cullin-RING ubiquitin ligase control, eif3m, unlike eif3e, is an essential gene required for global cellular protein synthesis and polysome formation. Using a ribonomic approach, this phenotypic distinction was correlated with a different set of mRNAs associated with the eIF3e and eIF3m complexes. Whereas the eIF3m complex appears to associate with the bulk of cellular mRNAs, the eIF3e complex associates with a far more restricted set. The microarray findings were independently corroborated for a random set of 14 mRNAs by RT-PCR analysis.

Conclusion: We propose that the PCI proteins eIF3e and eIF3m define distinct eIF3 complexes that may assist in the translation of different sets of mRNAs.

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Roles of eif3e and eif3min protein synthesis. (A) Subcellular localization. Live cells expressing GFP-tagged alleles of eif3b, eif3e, and eif3m at the endogenous genomic loci were examined by fluorescence microscopy. N-terminally GFP-tagged eif3f was expressed at low levels from the pREP81 plasmid. (B) Shut-off strains. Diploid heterozygous eif3f and eif3m deletion strains were transformed with pREP81 plasmids driving the thiamin-repressible expression of Myc-tagged eIF3f and eIF3m, respectively. Diploids were sporulated and homozygous disruptants carrying the eif3f and eif3m plasmids were selected. Cells were grown in liquid medium to an OD595 of 0.3 in the absence of thiamin, followed by promoter shut-off by the addition of thiamin. Samples were taken at the indicated time points after promoter shut-off and analyzed for the expression of plasmid borne eIF3f and eIF3m by immunoblotting with anti-Myc antibodies. (C) Effect on total protein synthesis. The eif3f and eif3m shut-off strains were maintained in the absence or presence of thiamin as indicated. Strains transformed with empty pREP81 plasmid were included as control. Cells were metabolically labeled with 35S-methionine, and aliquots of total cellular proteins were separated by SDS-PAGE and analyzed by autoradiography. The Coomassie Blue stained gel is shown to document equal protein loading. Data were quantified by PhosphorImager analysis and results are displayed in a bar graph. (D) Effect on polysomes. Polysome profiles were determined for the indicated strains as described in the Methods section. An eif3e deletion strain is shown for reference. (E) Effect on CSN-mediated regulation of cullin-RING ubiquitin ligases. Cul1p complexes were immunopurified from the indicated strains and employed in substrate-independent in vitro ubiquitylation reactions with purified E1, the E2 Cdc34p, ubiquitin, and ATP [26]. Polyubiquitin chains formed in the reaction are indicated (top panel). In contrast to csn5 mutants, derepression of Cul1p activity is not observed in cells lacking eif3f, eif3m, or eif3e. The neddylation state of Cul1p was determined by immunoblotting (middle panel). Hyperneddylated Cul1p only accumulates in csn5 mutants. Shut-off of eif3f and eif3m expression was verified by immunoblotting with anti-Myc antibodies (lower panel).
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Figure 2: Roles of eif3e and eif3min protein synthesis. (A) Subcellular localization. Live cells expressing GFP-tagged alleles of eif3b, eif3e, and eif3m at the endogenous genomic loci were examined by fluorescence microscopy. N-terminally GFP-tagged eif3f was expressed at low levels from the pREP81 plasmid. (B) Shut-off strains. Diploid heterozygous eif3f and eif3m deletion strains were transformed with pREP81 plasmids driving the thiamin-repressible expression of Myc-tagged eIF3f and eIF3m, respectively. Diploids were sporulated and homozygous disruptants carrying the eif3f and eif3m plasmids were selected. Cells were grown in liquid medium to an OD595 of 0.3 in the absence of thiamin, followed by promoter shut-off by the addition of thiamin. Samples were taken at the indicated time points after promoter shut-off and analyzed for the expression of plasmid borne eIF3f and eIF3m by immunoblotting with anti-Myc antibodies. (C) Effect on total protein synthesis. The eif3f and eif3m shut-off strains were maintained in the absence or presence of thiamin as indicated. Strains transformed with empty pREP81 plasmid were included as control. Cells were metabolically labeled with 35S-methionine, and aliquots of total cellular proteins were separated by SDS-PAGE and analyzed by autoradiography. The Coomassie Blue stained gel is shown to document equal protein loading. Data were quantified by PhosphorImager analysis and results are displayed in a bar graph. (D) Effect on polysomes. Polysome profiles were determined for the indicated strains as described in the Methods section. An eif3e deletion strain is shown for reference. (E) Effect on CSN-mediated regulation of cullin-RING ubiquitin ligases. Cul1p complexes were immunopurified from the indicated strains and employed in substrate-independent in vitro ubiquitylation reactions with purified E1, the E2 Cdc34p, ubiquitin, and ATP [26]. Polyubiquitin chains formed in the reaction are indicated (top panel). In contrast to csn5 mutants, derepression of Cul1p activity is not observed in cells lacking eif3f, eif3m, or eif3e. The neddylation state of Cul1p was determined by immunoblotting (middle panel). Hyperneddylated Cul1p only accumulates in csn5 mutants. Shut-off of eif3f and eif3m expression was verified by immunoblotting with anti-Myc antibodies (lower panel).

Mentions: Notably, no authentic CSN subunits were found in the Csn7Bp complex, consistent with the previous demonstration that S. pombe CSN contains only six non-essential subunits [13]. Structural analysis indicated that Csn6p is not only related to human CSN6, but shows a slightly greater overall similarity to human eIF3f (data not shown). Based on the functional characterization described below (Fig. 2), fission yeast Csn6p therefore appears to be the ortholog of human eIF3f.


PCI proteins eIF3e and eIF3m define distinct translation initiation factor 3 complexes.

Zhou C, Arslan F, Wee S, Krishnan S, Ivanov AR, Oliva A, Leatherwood J, Wolf DA - BMC Biol. (2005)

Roles of eif3e and eif3min protein synthesis. (A) Subcellular localization. Live cells expressing GFP-tagged alleles of eif3b, eif3e, and eif3m at the endogenous genomic loci were examined by fluorescence microscopy. N-terminally GFP-tagged eif3f was expressed at low levels from the pREP81 plasmid. (B) Shut-off strains. Diploid heterozygous eif3f and eif3m deletion strains were transformed with pREP81 plasmids driving the thiamin-repressible expression of Myc-tagged eIF3f and eIF3m, respectively. Diploids were sporulated and homozygous disruptants carrying the eif3f and eif3m plasmids were selected. Cells were grown in liquid medium to an OD595 of 0.3 in the absence of thiamin, followed by promoter shut-off by the addition of thiamin. Samples were taken at the indicated time points after promoter shut-off and analyzed for the expression of plasmid borne eIF3f and eIF3m by immunoblotting with anti-Myc antibodies. (C) Effect on total protein synthesis. The eif3f and eif3m shut-off strains were maintained in the absence or presence of thiamin as indicated. Strains transformed with empty pREP81 plasmid were included as control. Cells were metabolically labeled with 35S-methionine, and aliquots of total cellular proteins were separated by SDS-PAGE and analyzed by autoradiography. The Coomassie Blue stained gel is shown to document equal protein loading. Data were quantified by PhosphorImager analysis and results are displayed in a bar graph. (D) Effect on polysomes. Polysome profiles were determined for the indicated strains as described in the Methods section. An eif3e deletion strain is shown for reference. (E) Effect on CSN-mediated regulation of cullin-RING ubiquitin ligases. Cul1p complexes were immunopurified from the indicated strains and employed in substrate-independent in vitro ubiquitylation reactions with purified E1, the E2 Cdc34p, ubiquitin, and ATP [26]. Polyubiquitin chains formed in the reaction are indicated (top panel). In contrast to csn5 mutants, derepression of Cul1p activity is not observed in cells lacking eif3f, eif3m, or eif3e. The neddylation state of Cul1p was determined by immunoblotting (middle panel). Hyperneddylated Cul1p only accumulates in csn5 mutants. Shut-off of eif3f and eif3m expression was verified by immunoblotting with anti-Myc antibodies (lower panel).
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Figure 2: Roles of eif3e and eif3min protein synthesis. (A) Subcellular localization. Live cells expressing GFP-tagged alleles of eif3b, eif3e, and eif3m at the endogenous genomic loci were examined by fluorescence microscopy. N-terminally GFP-tagged eif3f was expressed at low levels from the pREP81 plasmid. (B) Shut-off strains. Diploid heterozygous eif3f and eif3m deletion strains were transformed with pREP81 plasmids driving the thiamin-repressible expression of Myc-tagged eIF3f and eIF3m, respectively. Diploids were sporulated and homozygous disruptants carrying the eif3f and eif3m plasmids were selected. Cells were grown in liquid medium to an OD595 of 0.3 in the absence of thiamin, followed by promoter shut-off by the addition of thiamin. Samples were taken at the indicated time points after promoter shut-off and analyzed for the expression of plasmid borne eIF3f and eIF3m by immunoblotting with anti-Myc antibodies. (C) Effect on total protein synthesis. The eif3f and eif3m shut-off strains were maintained in the absence or presence of thiamin as indicated. Strains transformed with empty pREP81 plasmid were included as control. Cells were metabolically labeled with 35S-methionine, and aliquots of total cellular proteins were separated by SDS-PAGE and analyzed by autoradiography. The Coomassie Blue stained gel is shown to document equal protein loading. Data were quantified by PhosphorImager analysis and results are displayed in a bar graph. (D) Effect on polysomes. Polysome profiles were determined for the indicated strains as described in the Methods section. An eif3e deletion strain is shown for reference. (E) Effect on CSN-mediated regulation of cullin-RING ubiquitin ligases. Cul1p complexes were immunopurified from the indicated strains and employed in substrate-independent in vitro ubiquitylation reactions with purified E1, the E2 Cdc34p, ubiquitin, and ATP [26]. Polyubiquitin chains formed in the reaction are indicated (top panel). In contrast to csn5 mutants, derepression of Cul1p activity is not observed in cells lacking eif3f, eif3m, or eif3e. The neddylation state of Cul1p was determined by immunoblotting (middle panel). Hyperneddylated Cul1p only accumulates in csn5 mutants. Shut-off of eif3f and eif3m expression was verified by immunoblotting with anti-Myc antibodies (lower panel).
Mentions: Notably, no authentic CSN subunits were found in the Csn7Bp complex, consistent with the previous demonstration that S. pombe CSN contains only six non-essential subunits [13]. Structural analysis indicated that Csn6p is not only related to human CSN6, but shows a slightly greater overall similarity to human eIF3f (data not shown). Based on the functional characterization described below (Fig. 2), fission yeast Csn6p therefore appears to be the ortholog of human eIF3f.

Bottom Line: Whereas neither eIF3e nor eIF3m contribute to the non-essential activities of CSN in cullin-RING ubiquitin ligase control, eif3m, unlike eif3e, is an essential gene required for global cellular protein synthesis and polysome formation.Using a ribonomic approach, this phenotypic distinction was correlated with a different set of mRNAs associated with the eIF3e and eIF3m complexes.We propose that the PCI proteins eIF3e and eIF3m define distinct eIF3 complexes that may assist in the translation of different sets of mRNAs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics and Complex Diseases, Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02115, USA. czhou@rics.bwh.harvard.edu

ABSTRACT

Background: PCI/MPN domain protein complexes comprise the 19S proteasome lid, the COP9 signalosome (CSN), and eukaryotic translation initiation factor 3 (eIF3). The eIF3 complex is thought to be composed of essential core subunits required for global protein synthesis and non-essential subunits that may modulate mRNA specificity. Interactions of unclear significance were reported between eIF3 subunits and PCI proteins contained in the CSN.

Results: Here, we report the unexpected finding that fission yeast has two distinct eIF3 complexes sharing common core subunits, but distinguished by the PCI proteins eIF3e and the novel eIF3m, which was previously annotated as a putative CSN subunit. Whereas neither eIF3e nor eIF3m contribute to the non-essential activities of CSN in cullin-RING ubiquitin ligase control, eif3m, unlike eif3e, is an essential gene required for global cellular protein synthesis and polysome formation. Using a ribonomic approach, this phenotypic distinction was correlated with a different set of mRNAs associated with the eIF3e and eIF3m complexes. Whereas the eIF3m complex appears to associate with the bulk of cellular mRNAs, the eIF3e complex associates with a far more restricted set. The microarray findings were independently corroborated for a random set of 14 mRNAs by RT-PCR analysis.

Conclusion: We propose that the PCI proteins eIF3e and eIF3m define distinct eIF3 complexes that may assist in the translation of different sets of mRNAs.

Show MeSH
Related in: MedlinePlus