UPF1 as the ultimate reaper: degrading mRNAs and proteins

Science Spotlight

UPF1 as the ultimate reaper: degrading mRNAs and proteins

From the Bradley Laboratory (Public Health and Basic Sciences Divisions)

Aug. 21, 2017

The information stored in DNA is transcribed into mRNAs, which are then translated into the proteins that make up the structure of the cell as well as influence its activity. This is known as the central dogma of molecular biology. In order to make sure that this process happens efficiently and properly, the cell has many ways of monitoring the progress and products of each step. One such mechanism degrades RNAs and is called nonsense-mediated decay (NMD) after its well-studied role in degrading abnormal mRNAs. However, it is estimated that 10-30% of normal human mRNAs are degraded by nonsense-mediated decay.1,2,3 Interestingly, previous research from the Bradley Lab (Public Health and Basic Sciences Divisions) found that NMD is compromised in cells overexpressing a gene known to cause facioscapulohumeral muscular dystrophy (FSHD).4 In addition to this, another study found that NMD efficiency and the levels of an essential NMD factor, UPF1, appear to decrease during the course of muscle cell development, called myogenesis.5 Given these observations, scientists in the Bradley Lab wondered whether the decrease in UPF1 and NMD efficiency was a cause or consequence of myogenesis. In a recent study published in Molecular Cell, they reported that expression of the NMD factor UPF1 does indeed control normal muscle cell development and through a surprising mechanism. They found that UPF1 controls the protein levels but not the mRNA of the master myogenic factor MYOD.

The scientists began by artificially altering the expression level of UPF1 to see if this altered muscle cell differentiation, or the molecular maturation of cells. To do this, they first "knocked-down" the expression of UPF1 in myoblasts (young muscle cells) by introducing small interfering RNAs (siRNAs) against UPF1 mRNAs. They found that cells where UPF1 expression was knocked-down expressed markers of muscle cell maturation much faster than wild-type myoblasts. Given this, they next performed experiments to test whether excess UPF1 would cause the opposite outcome: slowed myogenesis. They created myoblast cells where they could over-express UPF1 when the chemical doxycycline was added to the growth media. They found that when these cells were treated with doxycycline, they took longer to express markers of muscle cell differentiation.  Thus, their results demonstrate that UPF1 slows myogenesis.

diagram showing how UPF1 controls muscle cell differentiation by regulating the level of MYOD protein

UPF1 controls the speed of muscle cell differentiation by regulating the level of MYOD protein. (top row) In wild-type (WT) cells, UPF1 helps transfer ubiquitin (purple, Ub) marks to MYOD (yellow, MD) protein. MYOD marked with ubiquitins is then degraded by the proteasome (blue). With less MYOD around, the differentiation of muscle cells happens slowly. (bottom row) In cells where UPF1 is mutated, ubiquitins are not transferred to MYOD and therefore MYOD is not degraded and can induce muscle cell differentiation into myotubes (more mature cells) faster than in wild-type cells.

Image provided by Dr. Qing Feng (Bradley Lab)

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UPF1 is an essential regulator of RNA so the scientists hypothesized that UPF1 slows myogenesis by degrading the mRNAs of factors that promote myogenesis, such as MYOD. If that were true, they expected to find that knocking down UPF1 stabilized MYOD mRNAs and also that the excess MYOD protein appeared after the accumulation of MYOD mRNAs. To their surprise however, they did not find either prediction to be true. They measured MYOD mRNA stability in cells where they had knocked down UPF1 and did not find that the mRNAs were abnormally stable compared to control-treated cells. Secondly, the appearance of excess MYOD protein in cells where UPF1 had been knocked down preceded the appearance of excess MYOD mRNA by 15-18 hours. Therefore, UPF1 regulates MYOD protein levels independently of regulating its mRNA. 

The timing of MYOD protein stabilization when UPF1 was knocked down suggested that UPF1 directly stabilized MYOD protein. Additionally, previous studies had reported that UPF1 physically interacts with components of the ubiquitin-proteasome degradation machinery and that a part of the protein appears structurally similar to the essential RING domain of E3 ubiquitin ligases, enzymes that mark proteins with ubiquitins, which often targets them for degradation by the proteasome. Given these clues, scientists in the Bradley Lab wanted to test whether the RING-like domain of UPF1 was required for its activity in regulating MYOD protein. They mutated amino acids in the RING domain of UPF1 and first tested whether these mutations affected its role in RNA surveillance. They confirmed that three known RNA substrates of UPF1 in muscle cells were unaffected by the mutations. Given this, they next tested how the mutations to UPF1 affected its role in stabilizing MYOD protein. They found that MYOD protein was marked with ubiquitin, a mark that often precedes protein degradation, in cells with wild-type UPF1 but that these marks were dramatically reduced in cells expressing the RING-mutant UPF1. These results and others strongly suggest that UPF1 is involved in stabilizing MYOD protein, but not its mRNA, during muscle cell differentiation. 

Overall, this research uncovers that a single protein has distinct roles in mRNA and protein quality control. A connection between protein and mRNA stability has also been found in the ribosome-quality control complex, which degrades abnormal peptides but can also induce ribosome disassembly and degradation of the associated mRNA. Discovering such connections between molecular quality control machinery is not only exciting because it advances our understanding of normal cell function but also because it has implications for understanding mutations associated with disease. Indeed, downregulation of UPF1 was found in facioscapulohumeral muscular dystrophy and mutations to the UPF1 gene are found in several different cancers. Thus, understanding all of the functions carried out by core RNA processing factors such as UPF1 may improve our understanding and enable better treatment of certain diseases.

 

Feng Q, Jagannathan S, Bradley RK.  2017. "The RNA Surveillance Factor UPF1 Represses Myogenesis via Its E3 Ubiquitin Ligase Activity." Molecular Cell. 67, 239-251.

This research was supported by the Ellison Medical Foundation, the National Institutes of Health, and the FSH society.  

Additional citations:

1. Hurt JA, Robertson AD, Burge CB. 2013. "Global analyses of UPF1 binding and function reveal expanded scope of nonsense-mediated mRNA decay." Genome Research. 23, 1636-1650.

2. Lewis BP, Green RE, Brenner SE. 2003. "Evidence for the widespread coupling of alternative splicing and nonsense-mediated mRNA decay in humans. PNAS. 100, 189-192.

3. McIlwain DR, Pan Q, Reilly PT, Elia AJ, McCracken S, Wakeham AC, Ilte-Youten A, Blencowe BJ, Mak TW. 2010. "Smg1 is required for embryogenesis and regulates diverse genes via alternative splicing coupled to nonsense-mediated mRNA decay. PNAS. 107,12186-12191. 

4. Feng Q, Snider L, Jagannathan S, Tawil R, van der Maarel SM, Tapscott SJ, Bradley RK. 2015. "A feedback loop between nonsense-mediated decay and the retrogene DUX4 in facioscapulohumeral muscular dystrophy." Elife. 4, e04996.

5. Gong C, Kim YK, Woeller CF, Tang Y, Maquat LE. 2009. "SMD and NMD are competitive pathways that contribute to myogenesis: effects on PAX3 and myogenin mRNAs. Genes and Development. 23, 54-66.