Nonsense -mediated decay: A branched and regulated pathway
The nonsense-mediated mRNA decay (NMD) pathway is an RNA surveillance mechanism that rapidly degrades aberrant mRNAs with premature termination codons (PTCs). Recently, it has become apparent that NMD is also a posttranscriptional regulatory mechanism that regulates the expression of large subsets of normal transcripts. The physiological role of NMD in mammals is poorly understood, in part, because of the early embryonic lethality that results from complete knockout of NMD factors. To circumvent this problem, I elected to knockdown a NMD factor in Sertoli cells, a cell type not essential for mice survival that provides nutritional and functional support for male germ cells during spermatogenesis. Using a cell- and tissue-specific knockdown approach that depends on the microRNA (miRNA) pathway, I depleted the NMD factor UPF3B in mice and observed that these mice had only mild male reproductive defects. One explanation for these mild defects is that the factor that I chose to knockdown, UPF3B, is not essential for NMD. Indeed, I obtained evidence that UPF3B is required for only one branch of the NMD pathway and that many natural NMD mRNA substrates remain downregulated by NMD even when both UPF3B and its paralog, UPF3A, are strongly depleted in cell lines. A non-mutually exclusive explanation for why only a mild phenotype is elicited by UPF3B knockdown is that a buffering system exists that partially compensates for the loss of UPF3B. Indeed, I obtained evidence that seven NMD factors are regulated by negative feedback loops. When NMD is perturbed, the transcripts encoding these seven NMD factors are upregulated. At least some of the NMD factor transcripts are NMD substrates, explaining why they are feedback regulated. To study the physiological role of both the feedback regulatory loop and the UPF3B-dependent branch of the NMD pathway, I generated mice harboring an enhancer-trap mutation in the Upf3b gene. These Upf3b-mutant mice lack detectable Upf3b mRNA expression in all tissues and are viable and fertile. They will be examined in the future for cognitive defects, as humans with null mutations in UPF3B suffer from mental retardation.
Huang, Lulu, "Nonsense -mediated decay: A branched and regulated pathway" (2009). Texas Medical Center Dissertations (via ProQuest). AAI3420350.