If this story had come from a kitchen rather than a scientific journal, here are the ingredients of its recipe: an antibiotic used to prevent organ rejections, a pasta-like protein that comes in different lengths, and a nice fat soup bone. Blend them together and you get a new report from Achim Leutz's group that sheds light on how information that lies within genes in our DNA influences the amounts and types of proteins that are made from it. The resulting menus get served up to different tissues of our bodies with all kinds of effects: they determine whether our bones grow or shrink, when cells divide, how we respond to infections, and sometimes whether a tumor develops. The study, which appears in the Jan. 1, 2010 issue of Genes and Development, may shed light on bone diseases and potentially a range of other health problems.

Postdoc Klaus Wethmar and other members of Achim's lab have been working on several levels of a tricky biological problem at the same time. The most fundamental question they are asking is how cells use information in the genome to produce specific amounts and types of proteins. Correct regulation of protein production has a huge impact on every aspect of our lives, and is a central theme in searching for the causes of disease – the set of proteins that a cell produces often determines whether we're healthy or ill. More specifically, Achim's group has been working on processes in cells that contribute to bone diseases, and recently they showed how shorter and longer versions of a protein called C/EBP-beta control whether the body builds new bone or breaks it down.

As it turns out, this is a good starting point to tackle some more general questions about the cookbook of over 20,000 proteins encoded in the human genome. The DNA sequences of genes determine their ingredients, but these instructions are accompanied by extra information that tells cellular chefs how to prepare them to suit the tastes of different regions of the body. Reading the genome has given scientists a fairly encyclopedic knowledge of protein recipes, but some of the extra instructions have been hard to study in living organisms.

"The information in the genome contains genes which are transcribed into messenger RNAs," Achim says. "Part of such a messenger, called the main open reading frame (ORF), is then used as a template to be translated into protein. But a lot of the message does not encode the protein, and we are only beginning to understand the functions of some of these flanking regions."

Directly in front of the C/EBP-beta gene, for example, is a small bit of code that serves as an upstream ORF, or uORF (upstream simply means its comes in front of the protein-encoding region rather than in the middle of it, or behind it). Experiments have shown that such uORFs help determine the quantity of proteins made by cells, but these studies have been carried out in cell cultures and the test tube. The idea has been more difficult to test in animals, which would give a much clearer picture of the functions of such uORFs in humans. The fact that cells make several forms of C/EBP-beta has now given Klaus, Achim and their colleagues a handle on the question.

"The choice of longer or shorter forms of the molecule controls whether the body builds bone or breaks it down, and this can be observed by studying bone tissue under the microscope or in animals," Achim says. "We had the idea that the uORF of the C/EBP-beta gene might help control whether the protein gets made, and which form is produced. The animal models we are using could help us find out."

The researchers already knew that an antibiotic called rapamycin, which has such powerful effects on the immune system that it is used to block organ rejections, influenced which form of C/EBP-beta cells make. Previous studies showed that rapamycin works by influencing the activity of a protein called mTOR. That's interesting because one healthy function of mTOR is to sense changes in nutrition and other environmental factors, sending signals into cells that somehow trigger adjustments in their behavior. Generally, when mTOR activity is low, cells tune down the amount of proteins that they produce. In previous work, Achim and his colleagues have shown that this has an additional effect on C/EBP-beta. Cells make much less of the shorter form of the protein, which tips the balance in favor of the longer form, with some rather dramatic effects on bone and other tissue.

Recently Klaus and his colleagues introduced a mutation into the region of the uORF of C/EBP-beta in mice. This meant that this supposed regulatory code wouldn't be translated; the cell would simply ignore it. If the uORF was important, the mutation would likely disrupt normal production of the protein, hopefully with observable effects on bone and other tissues.

They discovered that the animals' cells continued to produce the longer form of C/EBP-beta protein, but they no longer produced enough of the shorter form. As a result, the body failed to make enough cells called osteoclasts, which play an important role in breaking down bone so that the tissue can heal and be renewed. The animals had unusually thick bones and problems with their livers, where the short protein form helps regenerate damaged tissue.

"This is the first time that the function of such a uORF in a living vertebrate animal has been demonstrated," Achim says. "It confirms our hypotheses that uORF elements have an effect on protein production. Additionally, in the case of C/EBP-beta, it influences the balance between which form of the protein gets made. That has implications in a range of processes, such as the development of bones and other tissues, regenerative processes, the disregulated growth of cells that leads to cancer, and the body's response to inflammations. Mutations in the uORFs other molecules have been clearly linked to human diseases. This shows that at least in some of these cases, the problem may lie with the process of translating them into proteins. And it gives us a much clearer idea of where to start in looking for potential therapies."

- Russ Hodge  

A. The bones of mice lacking the short form of C/EBP-beta (right) produce smaller and less efficient bone-breakdown cells called osteoclasts (red) than mice with both forms of the molecule (left). B. Precursor cells derived from the bone marrow of mutant mice (right) are less able to develop into osteoclasts (red) than those of normal mice (left).  

Reference:

Wethmar K, Bégay V, Smink JJ, Zaragoza K, Wiesenthal V, Dörken B, Calkhoven CF, Leutz A. C/EBPbetaDeltauORF mice--a genetic model for uORF-mediated translational control in mammals. Genes Dev. 2010 Jan 1;24(1):15-20.

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