Co-translational assembly of multiprotein complexes: a systems biology approach

The information to build a cell is carried in its DNA. To be used, a portion of DNA needs to be copied into another molecule called RNA, from which it can be 'translated' into a protein. Proteins are the components that directly build the cell and make it function. Proteins do not work on their own. They attach to each other to form complicated machines, sometimes made up of dozens of proteins. Groups of proteins that are bound to each other and work together are called protein complexes. Not much is known about how protein complexes are built by cells. One possibility is that proteins are first made, and then attach to each other to form a complex. A second possibility is that the proteins start to bind to each other while they are being made in the cell. There are a few known cases of protein complexes that are made in the second way. However, because detecting this phenomenon is very difficult, it is not known if cells usually choose this way of making protein complexes. I have recently developed methods that make it easy to tell if the formation of a complex proceeds in this way. The aim of this project is to apply this method to many protein complexes with very different functions, in order to understand how cells build protein complexes. Why is this important for a cell? There are several reasons. Proteins need to recognise each other to form protein complexes, and they can recognise each other because they have specific shapes that fit into each other (similar to a key fitting into a lock). The shape of a protein changes as it is being made, and it is possible that some proteins can no longer fit into each other once they are completely made. Also, some proteins may be toxic for the cell when they are free (but useful when they are part of the correct complex). A good way to avoid this is to put the proteins in the complex as soon as possible (even before they are finished). We will study these questions using simple yeast cells. Yeast cells are similar enough to us that what we can learn from them is useful to understand the human body. Because protein complexes are important for almost everything a cell does, we hope that understanding how they are made by cells will help understand how they function and what goes wrong with them during disease.

Most proteins perform their functions as part of stable multiprotein complexes. However, very little is known about how these complexes are formed in vivo. One possibility is that they are formed after their components have been fully translated (posttranslational assembly). An alternative is that protein-protein interactions are established as the proteins are translated (cotranslational assembly). Although there are a few examples of cotranslational assembly, there are no straightforward methods to detect this phenomenon. Therefore, it is unknown if this is a common pathway for the formation of multiprotein complexes. We have recently set up the methods to systematically identify RNAs associated with a protein. This is done by purifying RNA-protein complexes and identifying the RNAs using microarrays (RIp-chip, for Ribonucleoprotein Immunoprecipitation analysed with DNA chips). While using this method, we noticed that proteins frequently bind to RNAs that encode interacting proteins. This observation can be explained if the protein-protein interactions are formed as the proteins are translated, and the RNAs encoding them associate indirectly as part of a polysome. These results show that this approach can be used to systematically detect cotranslational assembly. The aim of this project is to investigate the hypothesis that cotranslational assembly is a widespread pathway for the formation of protein complexes. As a model we will use a simple eukaryotic organism, the fission yeast Schizosaccharomyces pombe. We will apply the RIp-chip method to a variety of proteins known or suspected to be part of multiprotein complexes. We will then concentrate on a smaller number of complexes and apply the same method to all the components, with the aim of understanding the assembly pathway of the complex. These experiments will reveal how general cotranslational assembly is, and whether it is preferentially used for certain types of protein complexes.