Functional roles of nonsense-mediated decay in a human muscular dystrophy and myogenesis

Abstract

Multiple layers of regulation occur in mRNA life cycle, from RNA processing to translation to degradation. These multi-step processes benefit the organism to achieve genomic complexity, while risking it with increasing likelihood that errors can be introduced along the way. To maintain mRNA fidelity, cells evoke quality control mechanisms, including nonsense-mediated decay (NMD), which degrades mRNAs undergoing prematurely terminated translation. While efficient degradation of undesired gene products seem necessary, NMD efficiency is highly variable between cell types, in diseased or healthy conditions, raising the question: Is this variation in NMD efficiency simply a consequence of, or an unknown contributor to the difference in cell phenotypes? To address this question, I compared variation in NMD in muscular dystrophy and myogenesis models, and identified previously unknown regulatory roles of NMD pathway/factor: 1. A feedback loop between compromised NMD and the disease gene DUX4 in a human muscular dystrophy (Feng et al., eLife, 2015). DUX4 is a transcription factor, whose mis-expression in skeletal muscle causes apoptosis and facioscapulohumeral muscular dystrophy (FSHD). In order to understand the mechanism(s) of DUX4 toxicity in muscle, we performed RNAseq analysis, and found DUX4 induces profound NMD inhibition. As a result of this, RNAs normally degraded by NMD accumulate in DUX4 expressing cells, including DUX4 mRNA itself. We thus found that inhibition of NMD by DUX4 protein stabilizes DUX4 mRNA through a double-negative feedback loop (Figure 1). We also found that DUX4 induces NMD inhibition by, at least partially, triggering proteolytic degradation of UPF1, a central component of the NMD machinery. In this study, we illustrated an unexpected mode of autoregulatory behavior of DUX4, with implications for FSHD pathogenesis, and identified a previously unknown mechanism of proteolytic regulation of the NMD pathway. 2. NMD factor UPF1 in regulating myogenesis via its E3 ubiquitin ligase activity (Feng et al., under review). During muscle differentiation, NMD efficiency also attenuates. In order to understand whether NMD plays a regulatory role in myogenesis, we genetically perturbed the level of UPF1, the key RNA helicase in mediating NMD. We found ectopically suppressing UPF1 accelerates myogenesis, while increasing UPF1 levels slows myogenesis. Surprisingly, we found UPF1 represses myogenesis by promoting the decay of MYOD protein, a transcription factor that is a master regulator of myogenesis, while leaving MYOD mRNA stability unaffected. Finally, we found UPF1 acts as an E3 ligase via its RING domain to promote MYOD protein ubiquitination and degradation (Figure 2). In this study, we characterized a regulatory role for UPF1 in myogenesis, and demonstrated that UPF1 provides a mechanistic link between the RNA and protein decay machineries in human cells. To sum up, in my thesis research, we found that NMD is a regulatory (rather than constitutive) mechanism, the magnitude of which can be tuned to regulate physiological or even pathological processes. Meanwhile, we identified that NMD can be tuned by proteasome-mediated proteolytic regulation, via controlling the turnover of NMD factor(s). Conversely, we found that UPF1, a key NMD component, can mediate protein degradation, by functioning as an E3 ubiquitin ligase. Together, our study suggests a potential functional interaction between RNA and protein quality control pathways, underlying the cellular consequences upon NMD variation.