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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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Analysis of csn6 and csn7b genes and proteins. (A) The csn6 and csn7b genes were disrupted in diploid S. pombe cells by inserting the ura4 marker. Diploids were sporulated and spore viability was examined by tetrad analysis. Only two spores were viable, indicating that csn6 and csn7b are essential. (B) Cell lysate from a strain carrying csn7b modified with five C-terminal pro-A tags and a TEV cleavage site at the endogenous genomic locus was absorbed to IgG resin, followed by elution of bound proteins with TEV protease (left panel) or SDS (right panel). Gels were stained with Coomassie Brilliant Blue, and proteins were identified by MALDI-TOF mass spectrometry. The asterisks denote degradation products of elF3c.
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Figure 1: Analysis of csn6 and csn7b genes and proteins. (A) The csn6 and csn7b genes were disrupted in diploid S. pombe cells by inserting the ura4 marker. Diploids were sporulated and spore viability was examined by tetrad analysis. Only two spores were viable, indicating that csn6 and csn7b are essential. (B) Cell lysate from a strain carrying csn7b modified with five C-terminal pro-A tags and a TEV cleavage site at the endogenous genomic locus was absorbed to IgG resin, followed by elution of bound proteins with TEV protease (left panel) or SDS (right panel). Gels were stained with Coomassie Brilliant Blue, and proteins were identified by MALDI-TOF mass spectrometry. The asterisks denote degradation products of elF3c.

Mentions: CSN complexes of higher eukaryotes typically contain eight distinct subunits (two MPN and six PCI proteins). However, in fission yeast only six subunits are known (Csn1p, 2p, 3p, 4p, 5p, 7Ap; Ref. [13]), none of which are essential for viability [40-42]. We noticed two genes in the Schizosaccharomyces pombe genome database, originally annotated as csn6 (SPBC4C3.07) and csn7b (SPAC1751.03), which encode MPN and PCI domain containing proteins with considerable similarity to metazoan CSN6 and CSN7B, respectively (data not shown). In order to determine whether these genes might function in the known biochemical pathways regulated by CSN, cullin deneddylation and Ubp12p-mediated deubiquitylation [24,26], we deleted csn6 and csn7b in wild-type diploid cells. Upon sporulation and tetrad analysis, only two viable spores could be recovered in each case, both of which were uracil auxotroph (Fig. 1A, and data not shown). Thus, unlike the genes encoding the six known subunits of the fission yeast CSN [41,42], csn6 and csn7b are essential.


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)

Analysis of csn6 and csn7b genes and proteins. (A) The csn6 and csn7b genes were disrupted in diploid S. pombe cells by inserting the ura4 marker. Diploids were sporulated and spore viability was examined by tetrad analysis. Only two spores were viable, indicating that csn6 and csn7b are essential. (B) Cell lysate from a strain carrying csn7b modified with five C-terminal pro-A tags and a TEV cleavage site at the endogenous genomic locus was absorbed to IgG resin, followed by elution of bound proteins with TEV protease (left panel) or SDS (right panel). Gels were stained with Coomassie Brilliant Blue, and proteins were identified by MALDI-TOF mass spectrometry. The asterisks denote degradation products of elF3c.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1173091&req=5

Figure 1: Analysis of csn6 and csn7b genes and proteins. (A) The csn6 and csn7b genes were disrupted in diploid S. pombe cells by inserting the ura4 marker. Diploids were sporulated and spore viability was examined by tetrad analysis. Only two spores were viable, indicating that csn6 and csn7b are essential. (B) Cell lysate from a strain carrying csn7b modified with five C-terminal pro-A tags and a TEV cleavage site at the endogenous genomic locus was absorbed to IgG resin, followed by elution of bound proteins with TEV protease (left panel) or SDS (right panel). Gels were stained with Coomassie Brilliant Blue, and proteins were identified by MALDI-TOF mass spectrometry. The asterisks denote degradation products of elF3c.
Mentions: CSN complexes of higher eukaryotes typically contain eight distinct subunits (two MPN and six PCI proteins). However, in fission yeast only six subunits are known (Csn1p, 2p, 3p, 4p, 5p, 7Ap; Ref. [13]), none of which are essential for viability [40-42]. We noticed two genes in the Schizosaccharomyces pombe genome database, originally annotated as csn6 (SPBC4C3.07) and csn7b (SPAC1751.03), which encode MPN and PCI domain containing proteins with considerable similarity to metazoan CSN6 and CSN7B, respectively (data not shown). In order to determine whether these genes might function in the known biochemical pathways regulated by CSN, cullin deneddylation and Ubp12p-mediated deubiquitylation [24,26], we deleted csn6 and csn7b in wild-type diploid cells. Upon sporulation and tetrad analysis, only two viable spores could be recovered in each case, both of which were uracil auxotroph (Fig. 1A, and data not shown). Thus, unlike the genes encoding the six known subunits of the fission yeast CSN [41,42], csn6 and csn7b are essential.

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