This competitive renewal extends our studies of nonsense-mediated mRNA decay (NMD) and its constituent factors and effectors in human and mouse cells and, since the last competitive renewal, using mice. NMD controls the quality of gene expression by preventing the production of potentially toxic proteins in health and in disease. It provides for the autoregulation of many RNA-binding proteins that regulate the splicing of the pre- mRNA from which they derive. NMD also promotes the adaptation of cells to changing environments utilizing the ~10% of cellular mRNAs that are natural NMD targets.
Aim 1 proposes to pursue our exciting finding that the abundance of NMD factors and the efficiency of NMD are upregulated in human neuronal cells deficient in Fragile X Mental Retardation Protein (FMRP). FMRP deficiency typifies Fragile X Syndrome (FXS), which is the most common single-gene cause of intellectual disability and autism. We will analyze the mechanistic and metabolic defects that typify the neuronal differentiation pathway of (i) SH-SY5Y neuroblastoma cells in which we have knocked-out the FMRP-encoding gene, FMR1, using CRISPR-Cas9n technology, and (ii) induced pluripotent stem cells (iPSCs) that we have generated from FXS-patient fibroblasts.
Aim 2 proposes to characterize the functional significance of our finding that cap-binding protein 80 (CBP80) directly binds the transcriptional co-activator PGC-1?. CBP80 is one subunit of the cap-binding heterodimer that is acquired co- transcriptionally during the expression of protein-encoding genes and, we have shown, persists through the pioneer round of translation, during which it functions in NMD. PGC-1? promotes mitochondrial energy- generating functions in response to physiological stresses that become bioenergetically sub-optimal during aging and disease. We will continue our molecular studies to determine how PGC-1? binding to CBP80 controls gene transcription using cultured myoblasts and also skeletal muscle regeneration in mice harboring five specific amino-acid changes in what we recently defined using crystallography and functional assays as the CBP80-binding motif of PGC-1?.
Aim 3 results from our successful genetic screen for new proteins influencing the efficiency of NMD using insertional gene-trap mutagenesis of haploid human cells that stably express an NMD reporter construct. We are validating proteins of special interest and are working to understand how each affects NMD. In summary, by satisfying previous aims, we have broadened the functional significance of NMD with the finding that NMD is hyper-activated in FMRP deficiency. We have broadened the functional significance of NMD factors with the finding that CBP80, which we have shown connects nuclear splicing to cytoplasmic translation via exon-junction complexes and the pioneer round of translation, also functions in earlier gene-expression steps via interactions with a transcriptional co-activator. We are also defining new effectors of NMD efficiency. Our results should lend new insight into disease- associated cellular metabolism.
Nonsense-mediated mRNA decay (NMD) is critical for human health. Either abnormally high or abnormally low levels of NMD factors are associated with intellectual disability and autism as well as defects in the generation of muscle fibers. This proposal aims to further examine how NMD factors regulate normal gene expression, how the efficiency of NMD contributes to normal cellular metabolism, and what goes wrong in cells when NMD factors or effectors are defective.
|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|
|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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