Understanding position-9 methylation of nucleocytosolic tRNA

The genetic information of every organism is stored in the DNA of each cell, and is expressed and decoded via RNA intermediates into protein. Both DNA and RNA use a code of 4 letters to store and express information. The four letters of RNA, A,G,C and U, are known since decades, but accumulating evidence shows that the cells do chemically modify these four letters to expand the decoding properties and function of RNA molecules. To date, more than a hundred different modifications have been identified, but only a handful of these have been intensively investigated. For the large majority of RNA modifications, we still have very little knowledge on how they are produced in the cell, and what is their actual function. In my project I will investigate two specific types of modification, found in a class of molecules called transfer RNAs (tRNAs). These modifications are found in a diverse range of species, including microorganism and humans, yet we do not know how many and which tRNAs have this specific modification. We have preliminary evidence that these modifications affect the shape of tRNAs, which is a key determinant of the function of the molecule. Remarkably, mutations in the genes of the enzymes that introduce these modifications cause a severe genetic disease, characterized by defects in brain development and diabetes. Using an innovative approach that allows us to take a snapshot of all the modified RNAs in a cell, we are now able to fully describe the landscape of tRNAs that have the modification of interest. We will characterize in detail the effect of these modifications on the tRNAs, and how they affect the shape of the molecule and its function. Moreover, we will study the enzymes that are responsible for attaching these modifications to tRNA, and we aim to clarify the effect on the physiology of cells when these modifications are missing. In a collaborative effort with national and international partner researchers, we aim to fully clarify the biology of this widespread tRNA modification type.

In the project titled "Understanding position-9 methylation of nucleocytosolic tRNA" carried out at the Center for Anatomy and Cell Biology of the Medical University of Vienna, we have investigated the biological function of two human proteins called TRMT10A and TRMT10B. TRMT10A and TRMT10B are two closely related enzymes that based on their sequence were expected to be involved in tRNA modification. tRNAs are adaptor molecules essential for the synthesis of proteins, and are chemically modified by a large number of modification enzymes. However, for many of those modifications the responsible enzymes are not known, and/or the actual effect of the modifications on the tRNA and their importance for the physiology of living organisms remains unclear. We have used genome engineered model cell cultures to study the two enzymes. Using biochemistry and modern analytical techniques including next generation sequencing, we have found that TRMT10A and TRMT10B have different targets and different mechanisms of action, as they introduce different type of chemical modifications in different target tRNAs. Specifically, TRMT10A modifies guanosine (G) to 1-methylguanosine (m1G), whereas TRMT10B modifies adenosine (A) to 1-methyladenosine (m1A), both at position 9 but of different tRNAs. These findings are surprising as TRMT10B is the first identified tRNA methyltransferase that is specific for m1A9 in eukaryotes. Furthermore, using molecular biology techniques and mass spectrometry we found that the lack of modification at position 9 causes alterations of the abundance and further modification of some tRNAs, possibly affecting their function in the cell. We also generated mutations in TRMT10A to resemble TRMT10B in the form of chimeric enzymes. With those experiments we have gained knowledge about how the two enzymes work, and we have identified specific amino acids of TRMT10A and TRMT10B which are involved in the different activities of the two enzymes and contribute to confer them their specificity.