The survival of cells depends on quality-control mechanisms that detect mistakes in gene expression. The subject of this proposal is nonsense-mediated decay (NMD), an RNA surveillance pathway that recognizes and degrades aberrant mRNAs that possess premature termination (nonsense) codons. T-cell receptor (TCR) and immunoglobutin (Ig) transcripts are frequent substrates of NMD, as they commonly acquire nonsense codons as a result of the programmed gene rearrangement events that occur during lymphocyte development to increase the repertoire of receptors on lymphocytes. Other NMD substrates are aberrant mRNAs transcribed from randomly generated mutant gen_s (with nonsense mutations and frameshift mutations) and those generated as a result of biosynthetic errors (mistakes during transcription and RNA splicing). Paradoxically, the downregulation of aberrant TCR and Ig transcripts occurs in the nuclearfraction of cells, yet the primary signal that triggers NMD (a nonsense codon) was until recently only known to be recognized by the translation apparatus in the cytoplasm. Further evidence that the nucleus has a role in this downregulatory response is that an intron downstream of a nonsense codon serves as the second signal to trigger NMD. TCR transcripts appear to have or to respond differently to this second signal, as they differ from all other known transcripts in the minimum distance between the nonsense codon and the downstream intron required to elicit NMD. In addition, TCR transcripts differ in some of the factors that bind downstream of a nonsense codon to trigger this second signal. TCR also has unique regulatory elements upstream of the nonsense codon that elicit much more robust downregulation (more than 20-fold) than all known transcripts from non-rearranging genes.
The Specific Aims of this application are (1) to characterize and understand the mechanism of the novel regulatory elements that act upstream of nonsense codons to cause robust downregulation of TCR transcripts, (2) to identify and characterize factors that act on the regulatory elements downstream of nonsense codons (exon-exon junctions or introns) to deliver the second signal essential for TCR NMD, and (3) to assess the physiological significance of NMD by generating and testing lymphocyte cell lines with a null mutation in the NMD gene UPF1. Understanding the physiological functions and mechanisms of NMD is important, as this RNA surveillance pathway is triggered in a variety of human genetic diseases and may protect individuals from the potentially deleterious truncated proteins generated as a result of nonsense and frameshift mutations.
| Goetz, Alexandra E; Wilkinson, Miles (2017) Stress and the nonsense-mediated RNA decay pathway. Cell Mol Life Sci 74:3509-3531 |
| Jones, Samantha H; Wilkinson, Miles (2017) RNA decay, evolution, and the testis. RNA Biol 14:146-155 |
| Borgmann, Jennifer; Tüttelmann, Frank; Dworniczak, Bernd et al. (2016) The human RHOX gene cluster: target genes and functional analysis of gene variants in infertile men. Hum Mol Genet : |
| Lou, Chih H; Shao, Ada; Shum, Eleen Y et al. (2014) Posttranscriptional control of the stem cell and neurogenic programs by the nonsense-mediated RNA decay pathway. Cell Rep 6:748-64 |
| Nguyen, Lam Son; Wilkinson, Miles F; Gecz, Jozef (2014) Nonsense-mediated mRNA decay: inter-individual variability and human disease. Neurosci Biobehav Rev 46 Pt 2:175-86 |
| Liu, Chen; Karam, Rachid; Zhou, YingQi et al. (2014) The UPF1 RNA surveillance gene is commonly mutated in pancreatic adenosquamous carcinoma. Nat Med 20:596-8 |
| Lasalde, Clarivel; Rivera, Andrea V; León, Alfredo J et al. (2014) Identification and functional analysis of novel phosphorylation sites in the RNA surveillance protein Upf1. Nucleic Acids Res 42:1916-29 |
| Karam, Rachid; Wengrod, Jordan; Gardner, Lawrence B et al. (2013) Regulation of nonsense-mediated mRNA decay: implications for physiology and disease. Biochim Biophys Acta 1829:624-33 |
| Ramaiah, Madhuvanthi; Shum, Eleen Y; Wilkinson, Miles F (2012) How to activate a gene: decap its associated noncoding RNA. Mol Cell 45:271-3 |
| Karam, Rachid; Wilkinson, Miles (2012) A conserved microRNA/NMD regulatory circuit controls gene expression. RNA Biol 9:22-6 |
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