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)
Type
Research Project (R01)
Project #
5R01GM059614-16
Application #
8708097
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Bender, Michael T
Project Start
Project End
Budget Start
Budget End
Support Year
16
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Rochester
Department
Biochemistry
Type
School of Medicine & Dentistry
DUNS #
City
Rochester
State
NY
Country
United States
Zip Code
14627
Popp, Maximilian Wei-Lin; Maquat, Lynne E (2014) Defective secretory-protein mRNAs take the RAPP. Trends Biochem Sci 39:154-6
Kurosaki, Tatsuaki; Li, Wencheng; Hoque, Mainul et al. (2014) A post-translational regulatory switch on UPF1 controls targeted mRNA degradation. Genes Dev 28:1900-16
Popp, Maximilian Wei-Lin; Maquat, Lynne E (2014) The dharma of nonsense-mediated mRNA decay in mammalian cells. Mol Cells 37:1-8
Trcek, Tatjana; Sato, Hanae; Singer, Robert H et al. (2013) Temporal and spatial characterization of nonsense-mediated mRNA decay. Genes Dev 27:541-51
Kurosaki, Tatsuaki; Maquat, Lynne E (2013) Rules that govern UPF1 binding to mRNA 3' UTRs. Proc Natl Acad Sci U S A 110:3357-62
Hwang, Jungwook; Maquat, Lynne E (2011) Nonsense-mediated mRNA decay (NMD) in animal embryogenesis: to die or not to die, that is the question. Curr Opin Genet Dev 21:422-30
Hwang, Jungwook; Sato, Hanae; Tang, Yalan et al. (2010) UPF1 association with the cap-binding protein, CBP80, promotes nonsense-mediated mRNA decay at two distinct steps. Mol Cell 39:396-409
Maquat, Lynne E; Tarn, Woan-Yuh; Isken, Olaf (2010) The pioneer round of translation: features and functions. Cell 142:368-74
Isken, Olaf; Maquat, Lynne E (2009) Telomeric RNAs as a novel player in telomeric integrity. F1000 Biol Rep 1:90
Schoenberg, Daniel R; Maquat, Lynne E (2009) Re-capping the message. Trends Biochem Sci 34:435-42

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