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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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Specific association of mRNAs with the eIF3e and eIF3m complexes. (A) The eIF3 complexes shown were affinity-purified as described in the Methods section, and separated by SDS-PAGE followed by staining with Coomassie Brilliant Blue. The labeled bands were identified by LC-MS/MS. eIF3f* refers to a band in the eIF3m complex that was not identified by mass spectrometry in this experiment, and is therefore assigned based on its comigration with eIF3f identified in the eIF3e complex (right lane). eIF3f was positively identified by MALDI-TOF mass spectrometry as a subunit of the eIF3m complex in Fig. 1B. (B) The indicated eIF3e- and eIF3m-associated mRNAs were extracted from the purified complexes and employed in RT-PCR reactions using primers specific for each mRNA (see Table 3). PCR products obtained with total S. pombe RNA are shown for reference in the left panel. The factor of enrichment of each mRNA in the respective eIF3 complex and the enrichment rank (out of all 106 mRNAs enriched in either complex) are indicated below the gels.
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Figure 5: Specific association of mRNAs with the eIF3e and eIF3m complexes. (A) The eIF3 complexes shown were affinity-purified as described in the Methods section, and separated by SDS-PAGE followed by staining with Coomassie Brilliant Blue. The labeled bands were identified by LC-MS/MS. eIF3f* refers to a band in the eIF3m complex that was not identified by mass spectrometry in this experiment, and is therefore assigned based on its comigration with eIF3f identified in the eIF3e complex (right lane). eIF3f was positively identified by MALDI-TOF mass spectrometry as a subunit of the eIF3m complex in Fig. 1B. (B) The indicated eIF3e- and eIF3m-associated mRNAs were extracted from the purified complexes and employed in RT-PCR reactions using primers specific for each mRNA (see Table 3). PCR products obtained with total S. pombe RNA are shown for reference in the left panel. The factor of enrichment of each mRNA in the respective eIF3 complex and the enrichment rank (out of all 106 mRNAs enriched in either complex) are indicated below the gels.

Mentions: Both eluates contained an identical set of proteins. In addition to Csn6p, the Csn7Bp complex contained the eIF3 core subunits eIF3a, b, c, and i, as well as the non-core subunit eIF3h (Fig. 1B, Table 1). The eIF3g core subunit was not detected using MALDI-TOF in this purification, but was detected in an independent purification of the Csn7Bp complex using the more sensitive method of nano-liquid chromatography and tandem mass spectrometry (LC-MS/MS, see Fig. 5A). However, eIF3e and eIF3d, two previously described components of S. pombe eIF3 [34,36,38,39] were consistently absent from the Csn7Bp complex when analyzed either by MALDI-TOF or by LC-MS/MS (Fig. 1B and 5A).


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)

Specific association of mRNAs with the eIF3e and eIF3m complexes. (A) The eIF3 complexes shown were affinity-purified as described in the Methods section, and separated by SDS-PAGE followed by staining with Coomassie Brilliant Blue. The labeled bands were identified by LC-MS/MS. eIF3f* refers to a band in the eIF3m complex that was not identified by mass spectrometry in this experiment, and is therefore assigned based on its comigration with eIF3f identified in the eIF3e complex (right lane). eIF3f was positively identified by MALDI-TOF mass spectrometry as a subunit of the eIF3m complex in Fig. 1B. (B) The indicated eIF3e- and eIF3m-associated mRNAs were extracted from the purified complexes and employed in RT-PCR reactions using primers specific for each mRNA (see Table 3). PCR products obtained with total S. pombe RNA are shown for reference in the left panel. The factor of enrichment of each mRNA in the respective eIF3 complex and the enrichment rank (out of all 106 mRNAs enriched in either complex) are indicated below the gels.
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Figure 5: Specific association of mRNAs with the eIF3e and eIF3m complexes. (A) The eIF3 complexes shown were affinity-purified as described in the Methods section, and separated by SDS-PAGE followed by staining with Coomassie Brilliant Blue. The labeled bands were identified by LC-MS/MS. eIF3f* refers to a band in the eIF3m complex that was not identified by mass spectrometry in this experiment, and is therefore assigned based on its comigration with eIF3f identified in the eIF3e complex (right lane). eIF3f was positively identified by MALDI-TOF mass spectrometry as a subunit of the eIF3m complex in Fig. 1B. (B) The indicated eIF3e- and eIF3m-associated mRNAs were extracted from the purified complexes and employed in RT-PCR reactions using primers specific for each mRNA (see Table 3). PCR products obtained with total S. pombe RNA are shown for reference in the left panel. The factor of enrichment of each mRNA in the respective eIF3 complex and the enrichment rank (out of all 106 mRNAs enriched in either complex) are indicated below the gels.
Mentions: Both eluates contained an identical set of proteins. In addition to Csn6p, the Csn7Bp complex contained the eIF3 core subunits eIF3a, b, c, and i, as well as the non-core subunit eIF3h (Fig. 1B, Table 1). The eIF3g core subunit was not detected using MALDI-TOF in this purification, but was detected in an independent purification of the Csn7Bp complex using the more sensitive method of nano-liquid chromatography and tandem mass spectrometry (LC-MS/MS, see Fig. 5A). However, eIF3e and eIF3d, two previously described components of S. pombe eIF3 [34,36,38,39] were consistently absent from the Csn7Bp complex when analyzed either by MALDI-TOF or by LC-MS/MS (Fig. 1B and 5A).

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