We aim to continue our studies of the pioneer translation initiation complex and the pioneer round of translation. Nonsense-mediated mRNA decay (NMD) is an important quality control mechanism that eliminates transcripts having the potential to generate truncated proteins that could be deleterious to cells. We have found that NMD in mammalian cells generally occurs when translation terminates more than -50- 55 nts upstream of a splicing-generated exon-exon junction during a pioneer round of translation. By definition, this round of translation utilizes mRNA that is bound by the cap binding protein heterodimer CBP80/20. The role of the exon-exon junction in NMD reflects the post-splicing deposition of an exon junction complex (EJC) of proteins that include the Upf NMD factors. We have found that EJCs typify spliced CBP80/20-bound mRNA but not its remodeled product, elF4E-bound mRNA. To date, we have shown that the pioneer translation initiation complex is functionally distinct from but structurally overlaps with the steady- state, i.e., elF4E-bound, translation initiation complex. We have identified degradative activities involved in NMD. We also began to unravel why some mRNAs are subject to nucleus-associated NMD whereas other mRNAs are subject to cytoplasmic NMD.
In Aim 1, we will study further the structure of the pioneer translation initiation complex before and during translation, and we will characterize the interacting domains of CBP80-Upf 1, CBP80-SMG1, CBP80-elF4GI, Upf 1 -eRF1 and Upf 1 -eRF3 that we have shown exist.
In Aim 2, we will determine why some mRNAs are targeted for nucleus-associated NMD and other mRNAs are targeted for cytoplasmic NMD and, in collaboration with Rob Singer's lab, we will localize the cellular site of nucleus-associated NMD using fluorescent in situ hybridization of single RNA molecules.
In Aim 3, we will solidify data obtained in collaboration with Ben Blencowe's lab indicating that there are functional differences among different EJCs or if any EJC that resides sufficiently downstream of a nonsense codon can trigger NMD. These differences will be exploited to determine if NMD is triggered by only the 3'-most EJC or if any EJC that resides more than 50-55 nucleotides downstream of a nonsense codon can trigger NMD. We expect that renewed support of this grant will allow us to continue to make major advances in understanding the mechanism of NMD in mammalian cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059614-12
Application #
7894531
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Bender, Michael T
Project Start
1999-08-01
Project End
2011-07-31
Budget Start
2010-08-01
Budget End
2011-07-31
Support Year
12
Fiscal Year
2010
Total Cost
$365,904
Indirect Cost
Name
University of Rochester
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
Kurosaki, Tatsuaki; Maquat, Lynne E (2018) Molecular autopsy provides evidence for widespread ribosome-phased mRNA fragmentation. Nat Struct Mol Biol 25:299-301
Cho, Hana; Rambout, Xavier; Gleghorn, Michael L et al. (2018) Transcriptional coactivator PGC-1? contains a novel CBP80-binding motif that orchestrates efficient target gene expression. Genes Dev 32:555-567
Kurosaki, Tatsuaki; Hoque, Mainul; Maquat, Lynne E (2018) Identifying Cellular Nonsense-Mediated mRNA Decay (NMD) Targets: Immunoprecipitation of Phosphorylated UPF1 Followed by RNA Sequencing (p-UPF1 RIP-Seq). Methods Mol Biol 1720:175-186
Walter, Nils G; Maquat, Lynne E (2018) Introduction-RNA: From Single Molecules to Medicine. Chem Rev 118:4117-4119
Popp, Maximilian W; Maquat, Lynne E (2018) Nonsense-mediated mRNA Decay and Cancer. Curr Opin Genet Dev 48:44-50
Popp, Maximilian W; Maquat, Lynne E (2016) Leveraging Rules of Nonsense-Mediated mRNA Decay for Genome Engineering and Personalized Medicine. Cell 165:1319-1322
Elbarbary, Reyad A; Maquat, Lynne E (2016) Coupling pre-mRNA splicing and 3' end formation to mRNA export: alternative ways to punch the nuclear export clock. Genes Dev 30:487-8
Kurosaki, Tatsuaki; Maquat, Lynne E (2016) Nonsense-mediated mRNA decay in humans at a glance. J Cell Sci 129:461-7
Maquat, Lynne E (2016) Eukaryotic antisense ahead of its time. Nat Rev Mol Cell Biol 17:204
Popp, Maximilian W; Maquat, Lynne E (2015) Attenuation of nonsense-mediated mRNA decay facilitates the response to chemotherapeutics. Nat Commun 6:6632

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