What happens in a cell – when it divides, how it specializes, and whether it wards off disease – largely depends on which proteins it makes. These molecules are translated from messenger RNAs, which are encoded in genes. But while most of a person’s cells contain a complete set of genes, no cell makes a complete set of proteins. Finding out how cells choose which molecules they produce is a major theme of today’s biology. The laboratories of Nikolaus Rajewsky and Matthias Selbach at the MDC have now obtained the deepest-ever look at one level of decision-making. For the first time, they have been able to measure and compare two ways that molecules called microRNAs control levels of proteins produced by cells. The study appears in the July 30 edition of the journal Nature.
For 15 years scientists have known that as well as encoding genes, DNA contains the recipes for the very small microRNAs. Their main function is to dock onto much larger messenger RNAs and prevent their translation into proteins. Nikolaus says this happens in two ways.
“Sometimes they trigger the messenger RNA’s destruction,” he says. “In other cases they allow it to survive, but block the protein-making machinery from getting access to it – which means that proteins don’t get made.” Observing this second process has been difficult, making it hard to determine when, where, and to what degree cells use the two types of control.
The scientists already had methods to find out which messenger RNAs are destroyed, by comparing cells that produce microRNAs to those that do not. If a messenger RNA disappears in the presence of a microRNA, it has probably been destroyed by it. The effect is more like a volume control than an on-off switch; microRNAs miss some of their targets. Not all of the messenger RNAs that slip through will be used to make proteins – microRNAs might still block the process of translation. But getting a look at this type of control has been hard.
Björn Schwannhäusser and Nadine Thierfelder, PhD students from the two labs, developed a way to do so. They labeled amino acids (the building blocks of proteins) with a stable, non-radioactive isotope and put them into cell cultures along with microRNAs. Now as new proteins were made, the cells plugged in the new forms of the amino acids. Mass spectrometry was used to take a survey of the cells’ proteins. Because the amino acid labeled with isotopes are heavier than their non-labeled counterparts, the difference can be detected through mass spectrometry. The study revealed changes in the abundance of particular proteins, accurately reflecting differences in the amounts produced by the cells. This permitted a comparison of the proteins made by cells with or without the microRNA. “This is the first method to measure global changes in protein synthesis,” Matthias says. “For the first time we can directly see how much of a protein is actually produced.“
Analyzing the results the researchers discovered that single microRNAs can reduce the protein output of hundreds of genes – but that the effect is mild. “We rarely saw more than a four-fold reduction in protein levels,” Matthias says. “Usually the effect was even smaller. It was like turning down the volume – a microRNA can tune down the production of a wide spectrum of proteins, but their levels don’t go all the way down to silence.”
The study provided several other insights into the function and biological relevance of microRNAs. The small molecules bind to small motifs in messenger RNAs called seeds. These can be either at the head or tail of an RNA molecule. “Docking onto the tail region seems to be more important to blocking translation,” Nikolaus says.
In parallel to measuring protein production, the researchers also surveyed messenger RNA levels. In this way they could see whether microRNAs mainly operate by destroying messenger RNA destruction or inhibiting their translation. Interestingly, the two types of control don’t always affect the same molecules. Some messenger RNAs are tuned down in both ways; other molecules are affected by only one type of microRNA control.
The study also provided some insights into the functions of microRNAs. “Some of these molecules are produced only in specific cells and tissues,” Nikolaus says. “Our study shows that if you force a ‘generic’ cell to produce one of these microRNAs, you see a global shift in protein production. The overall pattern becomes much more like that of the tissue where the microRNA is normally found. This suggests that microRNAs are important in pushing cells along particular pathways of specialization.”
- Russ Hodge
Selbach M, Schwanhäusser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N. Widespread changes in protein synthesis induced by microRNAs. Nature. 2008 Jul 30. [Epub ahead of print]