Eukaryotic termination results from the complex functional interplay between two release factors, eRF1 and eRF3, in which GTP hydrolysis by eRF3 couples codon recognition and peptidyl-tRNA hydrolysis by eRF1. Recycling of post-termination complexes (post-TCs) is promoted by the concerted action of ABCE1, an essential member of ATP-binding cassette family of proteins, and eRF1, which remains associated with post- TCs after peptide release. eRF1 thus participates in two successive stages of protein synthesis, termination and ribosome recycling. In some cases post-TCs do not undergo complete recycling, and termination is instead followed by reinitiation on the same mRNA, usually downstream of the stop codon. This mostly occurs after translation of short open reading frames (ORFs), whereas reinitiation after long ORFs is rare and appears to be limited to several virus families. Termination/recycling are also associated with two other important processes, mRNA surveillance by No-Go and nonsense-mediated decay (NGD and NMD, respectively). NGD targets mRNAs on which elongation complexes (ECs) are stalled by e.g. stable secondary structure. It is initiated by endonucleolytic cleavage in the vicinity of stalled ribosomes, which s stimulated by eRF1 and eRF3 paralogues Pelota and Hbs1 that together with ABCE1 can then dissociate ECs without prior peptide release. NMD targets aberrant mRNAs containing premature termination codons (PTCs), which are introduced by aberrant splicing, mutation or transcription errors. Recognition and targeting for degradation of PTC-containing mRNAs is mediated by conserved effectors, including UPF1 (a DEAD-box RNA helicase), UPF2, UPF3B and the UPF1 kinase SMG1. UPF1 interacts with eRF1, eRF3, UPF2, UPF3B and SMG1, and plays a key role in the process. It was suggested that ribosomes arrested at PTCs are recognized by a SURF (SMG1, UPF1, eRF1/3) surveillance complex, and that recruitment of UPF1 also impairs termination. The proposed studies will be based on the approach of in vitro reconstitution of all stages of protein synthesis (initiation, elongation, termination and ribosoma recycling) from individual purified translational components.
In Aim 1, we propose to investigate the structure of ribosome recycling and termination complexes corresponding to different stages in the process, and to characterize how termination is regulated by cis-acting mRNA elements and trans-acting factors.
In Aim 2 we will recapitulate different modes of reinitiation using our i vitro reconstituted system to determine their factor requirements and mechanisms.
In Aim 3 we will define the requirements for ABCE1/Pelota-mediated dissociation of stalled ribosomal elongation complexes, and will undertake an attempt to localize the site of endonucleolytic cleavage in NGD and to identify the responsible nuclease.
Aim 4 will be devoted to investigation of the influence of UPF1, depending on its phosphorylation status and the presence of UPF2 and UPF3 cofactors, on individual steps in the translation process.

Public Health Relevance

Mutations in the translation apparatus leading to defects in stop codon recognition and uORF-mediated translational control are implicated in genetic diseases, e.g. cystic fibrosis, muscular dystrophy, hereditary thrombocythemia and many others. About a third of all inherited human disorders are caused by mutations that introduce premature termination codons (PTC), which trigger mRNA degradation by nonsense-mediated decay (NMD). Clinically relevant antibiotics suppress PTC recognition, enhancing readthrough and ameliorating disease by partially restoring synthesis of full-length protein and impairing NMD, but the efficacy of suppression is variable: more detailed characterization of termination and NMD would facilitate further rational development of PTC suppression therapy.)

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM080623-06
Application #
8538426
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Bender, Michael T
Project Start
2007-05-01
Project End
2016-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
6
Fiscal Year
2013
Total Cost
$361,899
Indirect Cost
$135,003
Name
Suny Downstate Medical Center
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
040796328
City
Brooklyn
State
NY
Country
United States
Zip Code
11203
des Georges, Amedee; Hashem, Yaser; Unbehaun, Anett et al. (2014) Structure of the mammalian ribosomal pre-termination complex associated with eRF1.eRF3.GDPNP. Nucleic Acids Res 42:3409-18
Skabkin, Maxim A; Skabkina, Olga V; Hellen, Christopher U T et al. (2013) Reinitiation and other unconventional posttermination events during eukaryotic translation. Mol Cell 51:249-64
Jackson, Richard J; Hellen, Christopher U T; Pestova, Tatyana V (2012) Termination and post-termination events in eukaryotic translation. Adv Protein Chem Struct Biol 86:45-93
Pisareva, Vera P; Skabkin, Maxim A; Hellen, Christopher U T et al. (2011) Dissociation by Pelota, Hbs1 and ABCE1 of mammalian vacant 80S ribosomes and stalled elongation complexes. EMBO J 30:1804-17
Pisarev, Andrey V; Skabkin, Maxim A; Pisareva, Vera P et al. (2010) The role of ABCE1 in eukaryotic posttermination ribosomal recycling. Mol Cell 37:196-210
Skabkin, Maxim A; Skabkina, Olga V; Dhote, Vidya et al. (2010) Activities of Ligatin and MCT-1/DENR in eukaryotic translation initiation and ribosomal recycling. Genes Dev 24:1787-801
Jackson, Richard J; Hellen, Christopher U T; Pestova, Tatyana V (2010) The mechanism of eukaryotic translation initiation and principles of its regulation. Nat Rev Mol Cell Biol 11:113-27
Cheng, Zhihong; Saito, Kazuki; Pisarev, Andrey V et al. (2009) Structural insights into eRF3 and stop codon recognition by eRF1. Genes Dev 23:1106-18
Fan-Minogue, Hua; Du, Ming; Pisarev, Andrey V et al. (2008) Distinct eRF3 requirements suggest alternate eRF1 conformations mediate peptide release during eukaryotic translation termination. Mol Cell 30:599-609
Pisarev, Andrey V; Hellen, Christopher U T; Pestova, Tatyana V (2007) Recycling of eukaryotic posttermination ribosomal complexes. Cell 131:286-99