Ph. D. (1971) Brandeis University
Translation termination, NMD, and the development of therapeutic nonsense suppression
Unlike the other 61 triplets of the genetic code, the nonsense codons UAA, UAG, and UGA fail to encode amino acids and, instead, serve as translation termination signals. Nonsense codons generated by mutations, gene expression errors, or alternate splicing can inactivate gene function by promoting premature translational termination. The latter event leads to the production of only a truncated version of a protein and to mRNA destabilization by an mRNA quality control mechanism that we have dubbed nonsense-mediated mRNA decay (NMD). NMD is operative in essentially all eukaryotic cells examined and ensures that nonsense-containing mRNAs do not accumulate as substrates for the translation apparatus. In turn, the elimination of these transcripts prevents the accumulation of potentially toxic polypeptide fragments.
Using the yeast Saccharomyces cerevisiaeas a model system, much of the work in my lab is targeted to understanding the mechanistic details of NMD. Our experiments have led us to formulate the faux UTR model for NMD in yeast (see below), and independent studies in higher organisms have provided strong support for the general applicability of this model to all eukaryotes. Our data indicate that premature and normal termination differ mechanistically, with premature termination being a relatively inefficient process that leads to the ribosomal recruitment of at least three key regulatory proteins (Upf1, Upf2/Nmd2, and Upf3). These factors subsequently function in the dissociation of the premature termination complex and in the recruitment of the decapping enzyme responsible for initiating decay of the transcript. With the goal of further testing the faux UTR model, current studies in the lab are attempting to: a) define the timing and interaction-dependencies of Upf1, Upf2/Nmd2, and Upf3 association with prematurely terminating ribosomes, b) delineate the protein:protein interactions that link the decapping enzyme to the UPF/NMD factors, c) evaluate the functional differences of normal vs. premature termination, and d) characterize the Upf1 activity that dissociates and/or remodels ribosomal subunits at premature termination codons. To address these goals, we exploit yeast genetics, RNA biology, cell-free translation, and, in collaboration with the Moore (UMMS) and Gelles (Brandeis) labs, single-molecule analyses of protein synthesis. Additional studies in the yeast system seek to characterize the physiological significance of endogenous NMD substrates, as well as the mechanism by which some transcripts harboring premature terminators (e.g., cytoplasmic YRA1 pre-mRNA) can escape NMD.
In humans, nonsense mutations have been implicated in more than 2000 inherited diseases, including cystic fibrosis (CF), Duchenne muscular dystrophy (DMD), hemophilias, lysosomal storage disorders, skin disorders, and various cancers. A substantial fraction of the genetic disorders that arise from nonsense mutations are disabling or fatal and have only palliative treatment options at best. Given the large number of individuals that are collectively afflicted by nonsense mutations, a therapeutic approach to nonsense suppression could be of considerable medical benefit. Of particular importance is the possibility that a drug capable of suppressing nonsense in a given gene would also be capable of having the same effect on nonsense mutations in a completely different gene. Thus, under ideal circumstances a single drug could have the potential to treat hundreds, if not thousands, of different disorders where the only commonality would be their common origin from nonsense alleles. With this objective in mind, Dr. Stuart Peltz and I co-founded PTC Therapeutics Inc. (http://ptcbio.com/) in 1998, an endeavor that led to the identification and characterization of ataluren (PTC124), a novel, orally bioavailable small molecule that selectively promotes readthrough of premature nonsense codons (Welch et al., 2007 - see publications). Ataluren is currently being evaluated in clinical trials for several different nonsense-mediated genetic disorders (see: http://ptcbio.com/).
The faux UTR model. In normal termination (a), the ribosome harboring a nascent polypeptide approaches the UAG termination codon (top), engages the UAG codon in the ribosomal aminoacyl (A) site, binds the release factors eRF1 (Sup45) and eRF3 (Sup35), and releases the completed polypeptide (middle). The ribosome subunits are released from the mRNA and made available for another round of translation on either the same mRNA or another mRNA (bottom). Normal termination is thought to be highly efficient, possibly because interactions between Pab1 (the poly(A)-binding protein) and ribosome-associated eRF3 (Sup35) enhance the ability of eRF3 to stimulate the activity of eRF1 (Sup45). The spatial relationships of Pab1 and the termination site are exaggerated, but are meant to indicate that the effect of Pab1 depends on its proximity to the termination event. In premature termination (b), a ribosome harboring a nascent polypeptide approaches a premature UAG termination codon (top); it engages the UAG codon in its A site, binds eRF1 (Sup45) and eRF3 (Sup35), and fails to release the incomplete polypeptide (middle). Subsequently, the NMD factors Upf1, Nmd2/Upf2 and Upf3 bind to the release factors, stimulating peptide hydrolysis and 60S ribosomal subunit dissociation (bottom). The association of the NMD factors with the ribosome is postulated to facilitate the recruitment of the Dcp1-Dcp2 decapping enzyme complex to the mRNA, by virtue of the interaction of Dcp2 with Upf1, and to promote mRNA decapping. Premature termination is thought to be inefficient because the termination site lacks proximal Pab1 and/or other factors that are associated with a normal 3'-UTR. From: Amrani et al. Nature Reviews Molecular Cell Biology 7: 415-415 (2006).