We aim to continue our mammalian-cell studies that mechanistically characterize the pioneer round of translation and nonsense-mediated mRNA decay (NMD). We have found that pioneer translation initiation complexes consist of newly synthesized mRNAs that are bound at their 5'ends by the cap-binding protein (CBP) heterodimer CBP80-CBP20 and, if the mRNAs underwent splicing, at least one post-splicing exon-junction complex (EJC). Mammalian-cell mRNPs are targeted for NMD if they are bound by CBP80-CBP20, contain one or more EJCs that stably associate with some of the UPF NMD factors, and terminate translation in a way that triggers NMD. Steady-state translation initiation complexes, which we have shown derive from pioneer translation initiation complexes through translation-dependent and translation- independent mRNP remodeling steps, are bound at their 5'caps by eukaryotic translation initiation factor 4E (eIF4E) and lack detectable EJCs. Thus, steady-state translation initiation complexes, which support the bulk of cellular protein synthesis, are immune to NMD. While NMD is an important quality-control mechanism that typifies all eukaryotic cells, the restriction of NMD to newly synthesized mRNAs appears to be unique to mammalian cells.
In AIM 1, we will continue to study the structure and molecular rearrangements of mRNPs before, during and after the pioneer round of translation.
In AIM 2, we will further our collaboration with Rob Singer's lab to localize the cellular site of nucleus-associated NMD and, for comparison, the cellular site of cytoplasmic NMD using the fluorescent in situ hybridization of single RNA molecules. We will also use bimolecular fluorescence complementation coupled to Fvrster resonance energy transfer to localize within mammalian cells where particular trimolecular protein interactions that typify the pioneer round of translation and/or NMD occur.
In AIM 3, we will use methods that we are developing to identify which EJCs are functional during NMD. Notably, while EJC function depends on EJC position within the NMD target, data indicate that not all exon-exon junctions are associated with an EJC and there can be compositional and/or functional differences among different EJCs. We will examine both of these issues. Through each of the three aims, using tools and technologies that we have established over the past three decades, we expect to continue making significant advances toward understanding the mechanism of NMD in mammalian cells and defining protein-protein and protein-mRNA interactions that typify mammalian-cell mRNPs.

Public Health Relevance

Human gene regulation is monitored by the quality-control pathway called nonsense-mediated mRNA decay. This pathway eliminates the expression of potentially deleterious truncated proteins that are the consequence of routine mistakes made when cells express normal genes as well as the result of disease-associated mutations within genes. This proposal aims to further our studies of the molecular mechanism of nonsense-mediated mRNA decay.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059614-15
Application #
8516516
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Bender, Michael T
Project Start
1999-08-01
Project End
2015-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
15
Fiscal Year
2013
Total Cost
$376,058
Indirect Cost
$132,655
Name
University of Rochester
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
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
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; 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 (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
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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