Structure and Function of RNA Processing and Repair Enzymes

• Applicant: Dr. Jirka Peschek
• Subject Area: Biochemistry ; Structural Biology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 442512666

The cellular pool of ribonucleic acids (RNAs) is immensely diverse and complex, with numerous classes of RNAs functioning in a wide spectrum of biological processes. During their biosynthesis, RNA molecules undergo a vast number of co- and posttranscriptional processing and modification steps, which require dedicated enzyme machinery. One unique example of RNA processing is the protein-catalyzed non-conventional splicing of RNAs, which is an essential step during transfer RNA (tRNA) maturation. The splicing of intron-containing pre-tRNAs occurs in two steps: The intron is excised by a splicing endonuclease (SEN) and the resulting tRNA exon halves are ligated by tRNA ligase to form a matured tRNA. Eukaryotic tRNA ligases are not only key components of the cellular tRNA splicing machinery but also catalyse non-conventional mRNA splicing during the unfolded protein response, a major stress response pathway. In their enzymatic versatility lies the exciting potential for tRNA ligases as RNA repair enzymes. RNA can be subjected to damage through non-enzymatic hydrolysis or the action of endonucleases. These RNA cleavage events can be “sealed” by tRNA ligases, which catalyse the ligation of RNA molecules via phosphodiester bonds. With my proposal I aim to comprehend the structure and function of the eukaryotic tRNA splicing machinery and to uncover new RNA and ribonucleoprotein (RNP) repair pathways based on tRNA ligases. In my lab, we will use integrated structural biology approaches to obtain the first structures of eukaryotic SEN and tRNA ligase complexes with substrate RNA. To identify new RNA and RNP repair pathways, we will determine the cellular RNA substrates of tRNA ligases. To this end, we will identify new protein-RNA interactions and interconnected RNA processes using immunoprecipitation, sequencing-based approaches and biochemical reconstitution of tRNA ligase-based RNA repair systems. The mechanistic and structural insights into eukaryotic tRNA ligase complexes will synergize with the identified cellular roles in RNA and RNP repair. Together they will provide a comprehensive picture of how tRNA ligases function in RNA processing, quality control and repair. The proposed work aims at making fundamental discoveries in RNA biology and opening the door to pursue RNA processing enzymes as intervention points for human disease.

• DFG Programme: Independent Junior Research Groups
• Major Instrumentation: Liquid chromatography system
• Instrumentation Group: 1350 Flüssigkeits-Chromatographen (außer Aminosäureanalysatoren 317), Ionenaustauscher