30 May

The life and death of a single mRNA molecule

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A cellular system to get rid of faulty mRNAs was studied for the first time at the single molecule level. Researchers of the Tanenbaum group at the Hubrecht Institute used a new method to study this cellular quality control system, called nonsense-mediated mRNA decay, for individual mRNA molecules under the microscope in living cells. Their study was published in the scientific journal Molecular Cell on the 30th of May.

Schematic showing a ribosome translating an mRNA molecule, which results in the production of a complete protein.

From gene to protein
Even though cells in the body have different functions, each cell contains the same DNA. The different functions are determined by the genes in the DNA are active, and which are not. For active genes to perform their function, they are first copied into small molecules called mRNAs, which can leave the nucleus of a cell and go into the cytoplasm. Once in the cytoplasm, the mRNA molecules are translated into proteins by ribosomes, small mobile factories that can run along the mRNA molecule and read and translate the genetic code. The ribosome continues translating until it reaches a specific piece of code called a stop codon, that tells the ribosome that the protein is completed (see figure). Proteins are the workforce of the cell, and perform the functions that are encoded in the genes.

The process of going from DNA to protein can be compared to the construction of a car:  A blueprint of how to build the car exists in a document on the computer (the DNA in the nucleus). The blueprints are then printed (copied into mRNA molecules) and delivered to the factories (the ribosomes), where the car is assembled (translation of the mRNA into protein by the ribosome).

Nonsense-mediated mRNA decay
Mutations, or mistakes, in the DNA code of a gene can lead to the development of many different diseases. One type of mutation that often occurs in diseases is called a nonsense mutation, which creates a premature stop codon in a gene. Nonsense mutations result in shorter proteins, which are often toxic to the cell. In our analogy, this would be a car that can drive, but has no brakes or steering wheel.

Cells have a system to sense these mistakes and inhibit the production of these shorter, defective proteins. This system is called nonsense-mediated mRNA decay (NMD). In NMD, the ribosomes that are translating the mRNA can sense the mistake and mark the mRNA for destruction, just like the printed blueprints would be discarded to prevent the production of defective cars when the mistake is noticed.

A single mRNA molecule
Before, scientists used to look at an average of thousands or millions of mRNA molecules at the same time and then determine how these mRNAs were degraded. Although this can be informative, it provides limited insight into the complexity of NMD, just like an annual number of car crashes does not explain why individual cars crashed. Now, the researchers have developed a new method with which they can look at a single mRNA molecule to investigate what happens during NMD. This system can be used to address a plethora of questions, such as “Does the first ribosome that translates the mRNA sense the premature stop codon on an mRNA?” “What are the specific effects of NMD on the mRNA?”, “Do different subgroups of mRNA molecules exist that behave differently or that are more susceptible to NMD?” and “Do all cells have the same NMD efficiency?”.

Visualizing the life of an mRNA molecule
Using the newly developed method, the researchers can see 1) when each individual mRNA molecule is first synthesized, 2) the start of translation, and 3) the exact moment of mRNA destruction through NMD. Initially, the mRNA molecule is marked with a red fluorescent color. Once a ribosome starts translating the mRNA, the protein that is being made (and is still attached to the ribosome) is marked with a green fluorescent color. When NMD is activated, the mRNA molecule is cut in two pieces, resulting in the green and the red colored dot moving away from each other (see figure). This way, the researchers can follow single mRNA molecules during their entire lifetime in the cytoplasm and determine when, where and how the defective mRNAs are destroyed.

Upper part: Schematic of the way in which NMD is studied. Left: before the cleavage of an mRNA molecule, right: after the cleave of an mRNA molecule. Insets show what is seen under the microscope: mRNA as a red dot, protein as a green dot. When they overlap the mRNA has not yet been cleaved, when they move away from each other the mRNA has been cleaved. Bottom part: microscope images of mRNA molecules being translated. Translation of a normal mRNA molecule without a premature stop codon results in an overlapping red and green dot throughout the translation, while the translation of an mRNA with a premature stop codon results in the red and the green dot moving away from each other. Time is shown in min:sec.

New insights
The scientific community has long thought that the quality of an mRNA molecule was exclusively assessed immediately after the mRNA is produced, during the first round(s) of translation. Using their method, the researchers have been able to directly address this question. They found that most mRNA molecules with a premature stop codon are subject to NMD during their entire lifetime and that each ribosome that translates a defective mRNA has a similar chance of marking the mRNA for destruction. Furthermore, the researchers show that not all nonsense mutations are detected equally well by the quality control systems of a cell, and they reveal why certain defects are detected more efficiently than others.

Inhibiting NMD
Although NMD prevents production of toxic proteins, in some cases NMD can actually make diseases worse. When a car has only a small defect, such as a window that does not open, you may not want to get rid of the entire car. Similarly, some mutations result in proteins with only a small defect that could still be functional. However, since the mRNA is degraded by NMD, no protein is made, resulting in a more severe disease. Gaining insight into the mechanisms of NMD could help in developing therapies that specifically inhibit NMD, and treat patients in which NMD makes the disease more severe.


Publication
Single molecule imaging uncovers rules governing nonsense-mediated mRNA decay. Tim A. Hoek*, Deepak Khuperkar*, Rik G. H. Lindeboom, Stijn Sonneveld, Bram M. P. Verhagen, Sanne Boersma, Michiel Vermeulen and Marvin E. Tanenbaum. Molecular Cell 2019.
* contributed equally

For this research project, the Tanenbaum group collaborated with the Vermeulen group at the RIMLS in Nijmegen.

 

 

Marvin Tanenbaum is group leader at the Hubrecht Institute and Oncode Investigator.