Archease: a novel factor for tRNA splicing in human cells

The processing of tRNA precursors entails the removal of sequences at the 5` and 3` end as well as the excision of introns, in the process of tRNA splicing. Enzymatic activities required for the generation and ligation of tRNA exon halves have been identified in yeast and plants. Interestingly, animal cells use a different strategy at the ligation step in order to produce mature tRNAs. The actual tRNA ligase, however, has been elusive for almost thirty years. We have recently identified HSPC117 as the catalytic component of the human tRNA ligase (Popow et al, 2011). HSPC117 is present in human, flies, worms and, intriguingly, in Encephalitozoon cuniculi, a primitive cryptosporidyian parasite distantly related to fungi. Only eight types of proteins display this particular phyletic distribution and Archease, an uncharacterized, conserved protein is one of them. Here we reveal Archease as a new player in human tRNA splicing. RNAi-mediated depletion of Archease severely impairs the ligation of tRNA exon halves in cellular extracts, yet Archease is neither a ligase nor it interacts with the tRNA ligase complex. When added to a purified tRNA ligase complex, Archease functions as a product (mature tRNA)-release factor. We have also identified two extremely conserved residues that, when mutated, render Archease inactive. The finding of Archease, supporting the tRNA ligase in joining tRNA exon halves, revitalizes the field of non-canonical RNA splicing. In this proposal, we aim to understand how does Archease cooperate with the tRNA ligase complex in boosting tRNA ligation. Which interactions, protein-protein and RNA-protein, are essential for this process? How does the absence of Archease impact on the metabolism of tRNAs and other, still to be identified RNA molecules in vivo? We will also focus on other non-canonical RNA splicing processes where the tRNA ligase and Archease might be involved. Finally we will study the functional coupling between ligase and Archease not only in archaeabacteria, aiming for specific RNA substrates, but also in bacteria, which lack enzymatic tRNA splicing and yet encode ligase and archease for still unknown purposes.

The genetic information residing in the DNA is transcribed into messenger RNAs (mRNA) and then translated into proteins by the ribosome with the essential help of transfer RNAs (tRNA). The function of a tRNA is to read the mRNA and help the ribosome building a protein by adding a single aminoacid at a time. This FWF-Project focused on a particular aspect of the life of a tRNA molecule: its processing from a non-functional precursor to a functional molecule. After being produced tRNAs become chemically modified; some sequences are cleaved away by endonucleases and the remaining fragments joined by ligases. Proteins that cleave tRNAs are known. Ligase, however, have remained elusive. Back in 2011 we discovered the human tRNA ligase. More recently we found Archease as a co-factor of the ligase to increase its activity. This proposal aimed at studying different aspects of Archease. After three years of work we revealed the molecular mechanism used by Archease to activate the tRNA ligase. We also proved that together, tRNA ligase and Archease are essential for secretory cells to produce large amounts of proteins without colapsing. To learn about the in vivo functions of Archease we moved from cells to the mouse. We have therefore generated a mouse lacking the Archease gene which is currently being analyzed. The finding of Archease revealed an essential component in the processing oft RNA molecules and, at the same time, identified a potential target for diseases that rely on the ability of cells to handle the continuous production of large amounts of proteins.