The role of Ashwin in pre-tRNA processing and (m)RNA export

DNA molecules, assembled in chromosomes, constitute our genome, where the information to fully generate a living organism is stored. In our cells, a large fraction of the DNA is copied or transcribed into another molecule called RNA that the cell can read with perfection. However, right upon being transcribed, RNA molecules still lack functionality. Why? Because most RNAs need to be modified or extended or rearranged. In our laboratory, we are fascinated by enzymes that cut and re-join RNAs; we discovered many of those in our lab, here in Vienna. This proposal focuses on the physical, biochemical, and functional analysis of the enzyme that ligates glues pieces of transfer RNAs (tRNAs, essential to produce proteins) after removal of a short sequence named intron. If introns remain in mature tRNAs, then the tRNA will not be functional and the cell will die. The enzyme in charge of this reaction is called tRNA ligase and is composed of five different protein subunits. Only one can perform the chemical reaction. What do the other subunits do? This proposal deals with the least studied subunit of the tRNA ligase called Ashwin or ASW. Moritz Leitner, superb PhD student, has discovered that ASW plays two major roles within the tRNA ligase: a) transports it to the nucleus of the cell, where it executes the joining of tRNA molecules; b) links it to another protein complex called TREX (TRanscription EXport) that moves messenger RNAs RNA molecules that encode proteins to the cytoplasm, where proteins are generated. This proposal describes experiments to study those two functions of ASW. Regarding the transport to the nucleus, we speculate that ASW might use a very short sequence of amino acids as a signal to move the ligase to the nucleus. Here, we will change that sequence with the aim to abrogate that function. The second goal of our proposal is to visualize how ASW binds to TREX and whether such a link is functional. To visualize means to obtain the structure of the complex, i.e. ASW within the tRNA ligase (or alone) bound to TREX, taking advantage of cryo-electron microscopy and/or crystallization. In this regard, we will collaborate with Dr. Clemens Plaschka (IMP-Vienna), expert in the mRNA export pathway. On the functional side, we will evaluate the ability of ASW and the tRNA ligase to export (m)RNAs which bind to the tRNA ligase out of the nucleus. For this purpose, among other experiments, we will fractionate RNAs to distinguish the ones in the nucleus from those in the cytoplasm. If ASW binds specific RNA molecules and contacts TREX for their export, then removing ASW should lead to the accumulation of cytoplasmic RNAs inside the nucleus. In conclusion, we might unveil a potentially novel pathway to export RNAs bound by the tRNA ligase. Perspectives are exciting and we firmly believe that this proposal will reveal an important piece in the puzzle of gene expression in human cells.