pre-tRNA splicing: from enzymatic complexes to disease

The final step on the road of conveying genetic information, encrypted in chromosomal DNA, into proteins is called translation. At the core of this step are the so-called transfer-RNAs (tRNAs) molecules that serve in decoding this information. Some of them harbor short intervening sequences that have to be eliminated before tRNAs become fully functional. This is done in a process called pre-tRNA splicing, in which these sequences are initially removed by an enzymatic complex, the pre-tRNA splicing endonuclease, and the remaining sequences are subsequently joined by the enzymatic action of a tRNA ligase (tRNA ligation). In this proposal we will investigate whether the pre-tRNA splicing machinery regulates RNA molecules other than tRNAs, and reveal the precise function of each protein subunit within the human tRNA ligase complex by reconstituting the complex using recombinant proteins. We also aim at understanding disease mechanisms by obtaining the three-dimensional structure of the human pre-tRNA splicing endonuclease complex with mutations detected in patients suffering of a brain disorder, called pontocerebellar hypoplasia, and the structure of the human tRNA ligase complex to help in modeling small inhibitory molecules with potential in therapeutics. Our current work relies on another critical finding showing that pre-tRNA splicing in particular the ligation step is regulated by reactive oxygen species (ROS), which if present at high levels within the cell lead to biological imbalances and toxic effects (oxidative stress). To reveal the underlying mechanism we will take advantage of an analytical chemistry technique, called mass spectrometry, to identify crucial chemical modifications on the tRNA ligase complex impacting on its activity, and our collaborators in Japan will further test whether this phenomenon also takes place in Archaea and in yeast. This project is in essence multidisciplinary and relies on an intense collaboration with recognized structural biologists and experts in pre-tRNA processing in yeast and Archaea.

Genetic information is transmitted from the genomic DNA - our genes - through messenger RNA molecules (mRNA) to proteins, the working horses of the cell. However, there are RNA molecules other than mRNAs that have their own, independent functions. Among those, a very abundant class of RNAs are the transfer RNAs (tRNA), which are essential for the translation of mRNAs into proteins. tRNAs, like most cellular RNAs, undergo a series of modifications and transformations prior to become biologically active. My Laboratory focuses on a particular type of transformation, so-called "splicing", that entails the removal of specific stretches of RNA (introns) and the joining of the remaining RNA pieces (exons) by dedicated enzymes. In the project we have just carried out together with Dr. Simon Trowitzsch (University of Frankfurt) and Prof. Martin Jinek (University of Zurich), we revealed for the first time the three-dimensional structure of the two enzymes that execute the splicing of tRNA molecules: the tRNA splicing endonuclease (TSEN) complex, that removes the single intron in precursor tRNAs, and the enzyme that joins the remaining exons, known as the tRNA ligase complex. Despite the great progress during the course of the project, we unfortunately did not manage to obtain high-resolution structures of the entire enzymatic complexes because these enzymes are not easy to crystallize; they do not become rigid enough in order to form a crystal, and therefore the structure is difficult to determine. However, and in parallel with the structural determinations, we did make a large step in the biochemical analysis of these enzymes. Specifically, and with an eye on disease, we characterized the function of TSEN complexes with mutations leading to a rare neurological disease, known as pontocerebellar hypoplasia (PCH). Yet, why mutations in TSEN cause PCH remain largely unknown. We also studied a particular regulatory mechanism of the tRNA ligase complex, the enzyme that joins the two tRNA exons once the intron has been removed. Surprisingly, the tRNA ligase complex happens to be sensitive to molecules that arise under oxidative conditions; one of those molecules is hydrogen peroxide (H2O2). We identified an enzyme called PYROXD1, which co-evolved with the tRNA ligase to protect it from oxidation. In a paper that we have just published in the journal "Molecular Cell", we studied the "protector" in very much detail, both at the biochemical and structural levels, and dissected the (very novel) protection mechanism. Our studies on the structure and biochemical analysis of the TSEN complex and the tRNA ligase complex have been completed and will be submitted for publication shortly.