This proposal describes experiments to determine the mechanism and prevalence of a new translational initiation process. Translational initiation on most capped eukaryotic mRNAs begins with the recruitment of ribosomal 40S subunits to the 5' end of the mRNA via an interaction of ribosome-associated and cap binding protein complex-associated factors. The 40S subunit then scans the mRNA until an appropriate AUG state codon is encountered, at which a 60S ribosomal subunit joints to assemble a 80S ribosome, with the AUG start codon positioned in the ribosomal P-site. However, certain viral and cellular mRNAs have been identified that contain internal ribosome entry sites (IRES) elements that can direct 40S subunits directly to the mRNA. An IRES element from the intergenic region of critical paralysis virus (IGR-IRES_ directs the formation of initiating 80S ribosomes without initiator tRNAmet/eIF2- GTP complexes of GTP hydrolysis, and translation commences from the A-site of the ribosome, with RNA sequences in the IRES itself interacting at the P-site. Using ribosome assembly assays, toe-printing analysis and chemical and structural probing techniques, we will study the mechanisms by which ribosomal subunits are recruited to the IGR- IRES and by which 80S ribosomes are assembled on the IGR-IRES. The finding that the IGR-IRES is functioning under certain conditions in Saccharomyces cerevisiae opens the possibility to use both biochemical and genetic approaches to study the steps in IRES-mediated translation initiation complexes can be formed on the IGR-IRES when functional initiator tRNAmet/eIF2-GTP complexes are limiting in cultured cells. Finally, using cDNA microarray-based approaches, cellular mRNAs that display IGR-IRES like activities will be isolated and characterized. It is predicted that this unusual translation initiation pathway may operate when cells encounter a variety of stress situations, such as viral infection, heat shock, nutritional deprivation and hypoxia.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM055979-06
Application #
6626008
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Rhoades, Marcus M
Project Start
1996-09-01
Project End
2006-02-28
Budget Start
2003-03-01
Budget End
2004-02-29
Support Year
6
Fiscal Year
2003
Total Cost
$272,102
Indirect Cost
Name
Stanford University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Cevallos, Randal C; Sarnow, Peter (2005) Factor-independent assembly of elongation-competent ribosomes by an internal ribosome entry site located in an RNA virus that infects penaeid shrimp. J Virol 79:677-83
Cherry, Sara; Doukas, Tammy; Armknecht, Susan et al. (2005) Genome-wide RNAi screen reveals a specific sensitivity of IRES-containing RNA viruses to host translation inhibition. Genes Dev 19:445-52
Qin, Xiaoli; Sarnow, Peter (2004) Preferential translation of internal ribosome entry site-containing mRNAs during the mitotic cycle in mammalian cells. J Biol Chem 279:13721-8
Spahn, Christian M T; Jan, Eric; Mulder, Anke et al. (2004) Cryo-EM visualization of a viral internal ribosome entry site bound to human ribosomes: the IRES functions as an RNA-based translation factor. Cell 118:465-75
Thompson, Sunnie R; Sarnow, Peter (2003) Enterovirus 71 contains a type I IRES element that functions when eukaryotic initiation factor eIF4G is cleaved. Virology 315:259-66
Jan, Eric; Kinzy, Terri Goss; Sarnow, Peter (2003) Divergent tRNA-like element supports initiation, elongation, and termination of protein biosynthesis. Proc Natl Acad Sci U S A 100:15410-5
Sarnow, Peter (2003) Viral internal ribosome entry site elements: novel ribosome-RNA complexes and roles in viral pathogenesis. J Virol 77:2801-6
Bushell, Martin; Sarnow, Peter (2002) Hijacking the translation apparatus by RNA viruses. J Cell Biol 158:395-9
Fernandez, James; Yaman, Ibrahim; Sarnow, Peter et al. (2002) Regulation of internal ribosomal entry site-mediated translation by phosphorylation of the translation initiation factor eIF2alpha. J Biol Chem 277:19198-205
Jan, Eric; Sarnow, Peter (2002) Factorless ribosome assembly on the internal ribosome entry site of cricket paralysis virus. J Mol Biol 324:889-902

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