Coupled translation: a novel process for translation of two overlapping open reading frames in mRNA

Translation is the final stage of the most fundamental process in biology in which the genetic material of the organism is turned into proteins. During the process of translation; ribosomes (which are the cellular machines that build proteins) make the new protein by following a plan written in a molecule called mRNA. mRNA is a single stranded molecule made up from 4 different components (called nucleotides) which act like letters in our alphabet to make a code. Scientists have been studying the way the ribosome machine works in eukaryotes (higher organisms) for many years and thought that all proteins are made in the same way. Viruses are a group of microscopic organisms which infect and cause diseases in other organisms. During the infection process they must use the infected cells apparatus including ribosomes to make more new viruses. Viruses have been forced to develop new methods to survive and multiply faster before the infected cell can destroy it. These new methods include the manipulation of the cells ribosomes to do novel things which benefit the virus. Importantly, all new strategies used by the virus must be compatible with the workings of the cell to function. Interestingly, by studying these new strategies Scientists have seen that not only, do the viruses use them, but the cells also do. We are studying a virus called RSV which most individuals will have likely to have been infected with as a child and will have a good chance of being infected again in the future. The RSV virus also uses a novel mechanism to express a protein called M2-2. Here instead of just making one protein from a piece of mRNA two proteins are made. We are interested in working out how this is possible. We have shown that in order to make the second protein: M2-2 the first protein M2-1 must be made. This means the ribosomes must go in reverse as the coding sequence for the second protein overlaps with the coding sequence of the first. This was an important finding as ribosomes had not previously been shown to have this ability. Important regions within this piece of mRNA have been discovered that allow the second protein to be made. These regions appear to be present thought the coding sequence of M2-1 which is amazing because in molecular terms this is a large distance. If we change these regions by introducing mutations the amounts of the second protein made are reduced. One region which is important in this process has been shown to contain a lot of secondary structure. Secondary structure occurs where the nucleotides in different parts of the mRNA molecule can interact together, which can be thought of as like fitting two pieces of a jig-saw together. As more pieces are added the structure gets stronger. We will further characterise the important segments within M2-1 to investigate the role they have on M2-2 production. To do this we must first finish mapping the position of all of the important regions. In particular we focus on the region that contains a lot of secondary structure. Different types of mutations will be introduced in these regions to confirm there importance. We also hope to examine in more detail the components of the ribosome machine that make the second protein M2-2. Finally we hope that we can solve the structure (complete the jig-saw) of the mRNA molecule to provide a bigger picture on this unique mechanism of protein expression.

A novel process of translational control has been described for the pneumovirus mRNA which encodes the M2-1 protein. The mRNA also contains a second ORF which directs the synthesis of the M2-2 protein. Initiation of translation of the second, M2-2, ORF is coupled to termination of translation of the first ORF such that its translation is dependent on termination of translation of ORF1. The second ORF sequences play no role in the coupling process. The overlap region of the two ORFs alone is not sufficient for coupled translation to occur but require sequences throughout the entire first ORF. In particular a critical region is centred approximately 150 nucleotides upstream of the ORF 2 initiation codons is essential. This region contains a significant degree of secondary structure and its mutation significantly reduces coupled translation. We will investigate the nature and role(s) of the sequences upstream of the overlap region in coupling process using a deletion and mutagenesis approach. Initial attention will focus on the region of stable secondary structure. The requirement for specific initiation factors in the coupling process in vivo and in vitro will also be investigated using specific IRES sequences which have different eIF requirements. This data will be confirmed by using reticulocyte lysates depleted for specific initiation factors and UV cross-linking studies. Human mRNAs shown to contain overlapping ORFs will be studied for their capacity to carry out coupled translation. When the requirements for coupled translation of overlapping ORFs are more fully described the system will be used to couple heterologous genes in a controlled way. This will further define the requirements for coupled translation and will open up the possibility of exploitation the system for expression of other genes.