The cell’s two-pronged approach to mitigating genetic errors

From the Bradley Lab, Public Health Sciences and Basic Sciences Divisions

“An error doesn’t become a mistake until you refuse to correct it.” -Orlando Batista

The cells in our bodies are exquisitely refined instruments. Most days, in most of us, trillions of cells work together in near-perfect harmony to keep us healthy and functioning. But to err is more than just human, and even the most refined instrument will malfunction given enough opportunity. Cancer is among the most classic examples of cellular error. As our cells divide by the billions each day, rare errors are made in the copying of DNA, leading to small, usually insignificant mutations. But the wrong mutation in the wrong place can alter the function of a protein and set that cell on a path towards tumor formation. Fortunately, evolution is not blind to such risks, and has built into our cells mechanisms to correct, or at least to 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 in Life Science Alliance, postdoctoral fellow Dr. Dylan Udy and Dr. Robert Bradley, professor in Fred Hutch’s Public Health Sciences and Basic Sciences Divisions and McIlwain Family endowed chair in Data Science, discover that NMD is a more thorough and effectual mechanism than previously realized.

One form of potentially problematic genetic error is the nonsense mutation, which causes premature termination of protein synthesis. “[NMD] is a eukaryotic cellular surveillance system that acts to prevent the accumulation of potentially deleterious 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. Thus, some transcripts containing PTCs, termed NMD-insensitive transcripts, cannot be targeted for degradation, and it has generally been 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. Moreover, they explain, “studies in yeast indicated that protein levels can be reduced to a greater degree 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 precisely 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 NMD-mutant b-globin gene was fused to the luciferase gene (to allow for quantification of protein levels by measuring luminescence) and placed under control of a doxycycline-inducible promoter (to allow for external control of gene expression). The authors then used CRISPR to stably insert this construct into human HEK-293 cells. After isolating stable cell lines, then added doxycycline to initiate transgene expression followed by assessment of mRNA levels (via qRT-PCR) and protein levels (via luciferase luminescence).

The group first showed they could activate and measure NMD in this system – comparing a wild-type transgene to one containing a mutation that should be subject to NMD (deemed NMD+), they observed that NMD+ transcripts were degraded more rapidly, and were present at reduced levels, compared to wile-type transcripts. Next, they measured the effect of NMD on protein levels and came to a surprising conclusion. Surprisingly, NMD+ protein levels dropped more than could be accounted for by the observed reduction in mRNA. Moreover, even if the group inhibited NMD via RNAi knockdown of NMD factors, NMD+ protein levels were markedly reduced. Thus, the authors concluded, some mechanism in addition to mRNA degradation must be preventing the accumulation of NMD+ proteins. What could that mechanism be? “One potential mechanism is through increased degradation of proteins encoded by NMD-sensitive mRNAs,” the group mused. To test this theory, they used a drug to block translation and measured protein levels over the next several hours. While a small increase in degradation of NMD+ proteins was observed relative to wild-type, the authors concluded that this was insufficient to account for the decrease in NMD+ protein levels.

For now, the explanation for 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: “A big question these results raise are what mechanisms are cells using to limit the accumulation of proteins translated from NMD-sensitive transcripts? Other groups are already starting to tackle these questions, including identifying factors that repress translation of NMD-sensitive transcripts and ubiquitin ligases that target the 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 proteomics techniques.”

nmd graph
Quantification of RNA (red) and protein (green) levels for two NMD+ luciferase genes relative to controls (black), revealing that proteins levels decrease substantially more than RNA levels. Image provided by Dr. Dylan Udy

This work was supported by the National Institutes of Health, the Leukemia and Lympoma Society, the Mark Foundation for Cancer Research, the Paul G. Allen Frontiers Group, and the Department of Defense.

Fred Hutch/UW Cancer Consortium members Robert Bradley contributed to this work.

Udy DB, Bradley RK. Nonsense-mediated mRNA decay uses complementary mechanisms to suppress mRNA and protein accumulation. Life Sci Alliance. 2021 Dec 8;5(3):e202101217. doi: 10.26508/lsa.202101217. PMID: 34880103; PMCID: PMC8711849.