A two-pronged cell approach to mitigate genetic errors

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

The cells in our bodies are exquisitely refined tools. Most days, on most of us, trillions of cells work together in near perfect harmony to keep us healthy and functioning. But the error is more than just human, and even the most accurate tools will fail if given enough opportunity. Cancer is one of the most classic examples of cellular error. When our cells divide by the billions each day, rare errors occur in DNA transcription, resulting in small, usually insignificant mutations. But the wrong mutation in the wrong place can alter the function of the protein and set that cell on a path toward tumor formation. Fortunately, evolution does not ignore such risks, and has built in our cells mechanisms to correct, or at least reduce the consequences of such errors. One such mechanism is nonsensical mRNA decay (NMD), a process that limits the production of some mutant proteins. In a new article in Life Sciences Allianceand Postdoctoral Fellow Dr. Dylan O’Day and Dr. Robert BradleyFred Hutch, professor in the Departments of Public Health and Basic Sciences and the McIlwain Family Chair in Data Science, discovered that NMD is a more comprehensive and effective mechanism than previously recognized.

One form of possible genetic error is irrational mutation, which causes premature termination of protein synthesis. “[NMD] is a eukaryotic cellular monitoring system that blocks the accumulation of potentially harmful truncated proteins by targeting mRNAs with early termination codons (PTCs) for degradation.” But this monitoring system is imperfect – the PTC must be in the correct position within the RNA transcript to be recognized Thus, some transcripts containing PTCs, termed NMD-insensitive transcripts, cannot be targeted for degradation, and it was generally assumed that the presence of such transcripts would lead to an accumulation of the truncated protein.The authors note, however, that there was a lack of comparison. The direct quantification between mRNA and protein levels in NMD studies.Moreover, they explain, “studies in yeast have indicated that protein levels can be reduced by a greater degree than mRNA levels,” suggesting that mRNA degradation may not be the full story. in the process.

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

The group first showed that it could activate and quantify NMD in this system – comparing a wild-type mutated gene to one containing a mutation that must undergo NMD (considered NMD+), they noted that NMD+ transcripts were degraded more rapidly, and were present at reduced levels, compared to with wile scripts. Next, they measured the effect of NMD on protein levels and came to a surprising conclusion. Surprisingly, NMD+ protein levels decreased more than can be explained by the observed decrease in mRNA. Moreover, even if the group inhibited NMD via RNAi knockdown of NMD factors, NMD+ protein levels were significantly decreased. Thus, the authors concluded that some mechanisms in addition to mRNA degradation should prevent the accumulation of NMD+ proteins. What could this mechanism be? “One possible mechanism is through increased degradation of proteins encoded by NMD-sensitive mRNAs,” the team hoped. To test this theory, they used a drug to block translation and measured protein levels over the next several hours. While a slight increase in NMD+ proteolysis was observed relative to the wild type, the authors concluded that this was not sufficient to explain the decrease in NMD+ protein levels.

Currently, the explanation for how NMD+ protein levels are regulated independently of mRNA degradation remains elusive. Unraveling this mystery is a major goal of Dr. Udi moving forward: “The big question these findings raise is what mechanisms do cells use to limit the accumulation of proteins localized from NMD-sensitive transcripts? Other groups have already begun to address these questions, including identifying factors that inhibit 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 NMD-sensitive endogenous transcripts, which may become easier to quantify in the future using more sensitive proteomic techniques.”