Immunochemical characterization of mammalian protein synthesis initiation factors

Meyer, Laurence; Milburn, Susan C.; Hershey, John W.B.

doi:10.1021/bi00261a003

Cited by 70 publications

(45 citation statements)

References 25 publications

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“…2, 3; data not shown). This suggests that any differences in the binding affinities of mutant IRESs for the 40S subunit are relatively small and not significant enough to cause the observed lack of translation activity in rabbit reticulocyte lysate reactions, which we estimate to contain z600 nM 40S subunits (Meyer et al 1982).…”

Section: The Pseudoknot As a Drug Targetmentioning

confidence: 88%

The HCV IRES pseudoknot positions the initiation codon on the 40S ribosomal subunit

Berry

Waghray²,

Doudna³

2010

RNA

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The hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) in its 59 untranslated region, the structure of which is essential for viral protein translation. The IRES includes a predicted pseudoknot interaction near the AUG start codon, but the results of previous studies of its structure have been conflicting. Using mutational analysis coupled with activity and functional assays, we verified the importance of pseudoknot base pairings for IRES-mediated translation and, using 35 mutants, conducted a comprehensive study of the structural tolerance and functional contributions of the pseudoknot. Ribosomal toeprinting experiments show that the entirety of the pseudoknot element positions the initiation codon in the mRNA binding cleft of the 40S ribosomal subunit. Optimal spacing between the pseudoknot and the start site AUG resembles that between the Shine-Dalgarno sequence and the initiation codon in bacterial mRNAs. Finally, we validated the HCV IRES pseudoknot as a potential drug target using antisense 29-OMe oligonucleotides.

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Section: The Pseudoknot As a Drug Targetmentioning

confidence: 88%

The HCV IRES pseudoknot positions the initiation codon on the 40S ribosomal subunit

Berry

Waghray²,

Doudna³

2010

RNA

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“…10-µg proteins from lung cancer cell line H1299 and normal lung fibroblast IMR90 were subjected to Western blot analysis. Antibodies used are AbD, AbF (Johnson et al, 1997), M116 (Johnson et al, 1997), and eIF3 antiserum (Meyer et al, 1982) A1 A2 A3 A4 A5 A6 A7 A8 B1 B2 B3 B4 B5 B6 B7 C1 C2 C3 C4 C5 C6 C7 C8 D1 D2 D3 D4 D5 D6 D7 D8 E1 E2 E3 E4 E5 E6 E7 E8 F1 F2 F3 F4 G1 G2 G3 G4 G5 G6 G7 Tissue Assignment Nevertheless, both Bachmann's and our studies suggest that p150/p170 over-expression correlates with cancers in human breast, cervix, esophagus and lung. In this study, we found no correlation between p170 overexpression and clinical parameters such as age, histology, and treatment history of the lung cancer patients tested.…”

Section: Discussionmentioning

confidence: 99%

“…Thirdly, eIF3 p170 cDNA (Johnson et al, 1997) was translated in rabbit reticulocyte lysate (RRL) and the translation product of 170-kDa reacted strongly with AbD on Western blot ( Figure 5D, lanes 1 with 2-5) and by immunoprecipitation (data not shown). Lastly, the higher level of p170 was detected in human lung cancer cell line H1299 than in normal lung fibroblast IMR90 by eIF3 authentic antibodies AbF (Johnson et al, 1997), M116 (Johnson et al, 1997), and eIF3 antiserum (Meyer et al, 1982).…”

Section: Cloning Of P170mentioning

confidence: 97%

Identification of a 170-kDa protein over-expressed in lung cancers

2001

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Summary Lung cancer is the leading cause for cancer death in both male and female populations. Although many molecular markers for lung cancer have been developed and useful for early detection of lung cancer, their function remains unknown. In this paper, we report our findings that a 170-kDa protein (p170) is over-expressed in all types of human lung cancers compared with normal tissues and it is identified as a subunit of translation initiation factor eIF3 by cDNA cloning. Translation initiation factors are a family of proteins that promote the initiation step of protein synthesis and are regulators of cell growth at the translational level. Further studies showed that p170 mRNA is ubiquitously expressed with higher levels in adult proliferating tissues (e.g. bone marrow) and tissues during development (e.g. fetal tissues). This study suggests that p170 and eIF3 may be important factors for cell growth, development, and tumorigenesis.

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“…eIF3 antibody (Meyer et al, 1982) and eIF3f antibody (Shi et al, 2003) were raised in a goat as described. Goat polyclonal eIF3b and p-eIF2a antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).…”

Section: Cell-free Translation Inhibition Assaymentioning

confidence: 99%

Decreased expression of eukaryotic initiation factor 3f deregulates translation and apoptosis in tumor cells

et al. 2006

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The eukaryotic initiation factor 3f (eIF3f) is the p47 subunit of the multi-subunit eIF3 complex. eIF3 plays an important role in translation initiation. In the present study, we investigate the biological function of eIF3f in translation and apoptosis in tumor cells. We demonstrated for the first time that eIF3f is downregulated in most human tumors using a cancer profiling array and confirmed by real-time reverse transcription PCR in melanoma and pancreatic cancer. Overexpression of eIF3f inhibits cell proliferation and induces apoptosis in melanoma and pancreatic cancer cells. Silencing of eIF3f protects melanoma cells from apoptosis. We further investigated the biological function of eIF3f. In vitro translation studies indicate that eIF3f is a negative regulator of translation and that the region between amino acids 170 and 248 of eIF3f is required for its translation regulatory function. Ectopic expression of eIF3f inhibits translation and overall cellular protein synthesis. Ribosome profile and ribosomal RNA (rRNA) fragmentation assays revealed that eIF3f reduces ribosomes, which may be associated with rRNA degradation. We propose that eIF3f may play a role in ribosome degradation during apoptosis. These data provide critical insights into the cellular function of eIF3f and in linking translation initiation and apoptosis.

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Immunochemical characterization of mammalian protein synthesis initiation factors

Cited by 70 publications

References 25 publications

The HCV IRES pseudoknot positions the initiation codon on the 40S ribosomal subunit

The HCV IRES pseudoknot positions the initiation codon on the 40S ribosomal subunit

Identification of a 170-kDa protein over-expressed in lung cancers

Decreased expression of eukaryotic initiation factor 3f deregulates translation and apoptosis in tumor cells

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