The dual cell approach to mitigating genetic errors

“An error does not become an error until you refuse to correct it.” -Orlando Batista

The cells of our body are exquisitely refined instruments. On most days, in most of us, trillions of cells work together in almost perfect harmony to keep us healthy and functioning. But to err is more than human, and even the most refined instrument will fail given enough opportunity. Cancer is one of the most classic examples of cellular error. As our cells divide by the billions each day, rare mistakes are made in DNA copying, leading to small, usually insignificant, mutations. But the wrong mutation in the wrong place can disrupt a protein’s function and put that cell on the path to tumor formation. Fortunately, evolution is not blind to such risks and has built mechanisms into our cells to correct, or at least limit, the consequences of such errors. One such mechanism is nonsense-mediated mRNA decay (NMD), a process that limits the production of certain mutant proteins. In a new article on Life Sciences Alliancepostdoctoral fellow Dr. Dylan Udy and Dr Robert Bradleyprofessor in the Fred Hutch Divisions of Public Health Sciences and Basic Sciences and chair of the McIlwain Family in Data Science, finds that NMD is a more comprehensive and effective mechanism than previously believed.

One potentially troublesome form of genetic error is the nonsense mutation, which causes premature termination of protein synthesis. β€œ[NMD] it is a eukaryotic cellular surveillance system that acts to prevent the accumulation of potentially harmful truncated proteins by targeting mRNAs with premature termination codons (PTCs) for degradation,” the authors explain. But this surveillance system is imperfect: a PTC must be in the correct position within an RNA transcript to be recognized. Therefore, some PTC-containing transcripts, termed NMD-insensitive transcripts, cannot be subject to degradation, and it has been generally assumed that the presence of such transcripts would lead to the accumulation of truncated protein. The authors note, however, that there has been a lack of direct quantitative comparison between mRNA and protein levels in NMD studies. Furthermore, they explain, “studies in yeast indicated that protein levels can be reduced to a greater extent than mRNA levels,” suggesting that mRNA degradation may not be the whole story in this process.

The authors’ goal was to establish a reporter system to accurately quantify mRNA and protein levels in human cells carrying nonsense mutations. To this end, they generated a new genetic element in which the wild-type or mutant b-globin gene in NMD was fused with the luciferase gene (to allow quantification of protein levels by luminescence measurement). and placed under the control of a doxycycline-inducible promoter (to allow external control of gene expression). The authors then used CRISPR to stably insert this construct into human HEK-293 cells. After stable cell lines were isolated, doxycycline was added to initiate transgene expression, followed by assessment of mRNA levels (via qRT-PCR) and protein levels (via luciferase luminescence).

First, the group showed that they could activate and measure NMD in this system; comparing a wild-type transgene with one containing a mutation that should be subject to NMD (considered NMD+), they found that NMD+ transcripts were degraded more rapidly and were present at reduced levels, compared to wile-type transcripts. They then measured the effect of NMD on protein levels and came to a surprising conclusion. Surprisingly, NMD+ protein levels fell more than could be explained by the observed reduction in mRNA. Furthermore, even if the group inhibited NMD through RNAi removal of NMD factors, NMD+ protein levels were markedly reduced. Therefore, the authors concluded, some mechanism other than mRNA degradation must prevent the accumulation of NMD+ proteins. What could that mechanism be? “One potential mechanism is through increased degradation of NMD-sensitive mRNA-encoded proteins,” the group mused. To test this theory, they used a drug to block translation and measured protein levels over the next few hours. Although a small increase in NMD+ protein degradation was observed relative to wild-type, the authors concluded that this was insufficient to explain the decrease in NMD+ protein levels.

For now, the explanation of how NMD+ protein levels can be regulated independently of mRNA degradation remains elusive. Unraveling this mystery is a key goal for Dr. Udy going forward: β€œA big question raised by these results is what mechanisms cells use to limit the accumulation of translated proteins from NMD-responsive transcripts. Other groups are already beginning to address these questions, including identifying factors that repress translation of NMD-sensitive transcripts and ubiquitin ligases that target truncated proteins for degradation. We are also very interested in the protein:mRNA ratios of endogenous NMD-sensitive transcripts, which may become easier to measure in the future with more sensitive proteomic techniques.”

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