Caractérisation in silico et purification des ligases à ARN de type RtcB de Diplonema papillatum
Ribonucleic acids (RNA) undergo several post-transcriptional modifications before fulfilling their role in the cell. One of the actors responsible for these modifications are RNA ligases. There are two main families of RNA ligases, namely “ATP-grasp” and “RtcB-like”. Despite the fact that these enzymes have similar biological roles in the cell, their molecular mechanisms are very different. Our team has discovered in a marine eukaryote the presence of three genes encoding RtcB RNA ligase homologs. This is unusual considering that eukaryotes generally have only one gene encoding for an homolog of this ligase. Diplonema papillatum, the organism in question, is a eukaryote that has grown in popularity in recent years due to the particular mode of expression of its mitochondrial genetic material. Its genome is fragmented into several pieces called modules which, during the RNA maturation process, these modules undergo ligation via trans-splicing. We posit that one of the RtcB-type ligases present in D. papillatum, more specifically DpRTCB1, is a major player in this trans-splicing phenomenon. We also believe that the other two RtcB ligases present in this organism, DpRTCB2 and DpRTCB3, each have its own role in the cell. We therefore established in silico models for these proteins which could hint at the cofactors required by these three enzymes. We also propose a classification system for RtcB-type ligases according to their biological roles and variations in known active site residues. We attempted to purify DpRTCB fusion proteins for future enzymatic assays in order to get a better understanding in the biological role of these enzymes. These proteins, however, formed inclusion bodies making their purification difficult. Thus, we demonstrate various techniques that currently exist to attempt to purify proteins from insoluble aggregates. This Master’s thesis attempts to predict the potential cofactors and substrates necessary for future biochemical assays of DpRTCB enzymes. We also establish a robust foundation for a classification system of RtcB-type ligases. Among other things, this document provides basic solutions to researchers wishing to purify proteins that form inclusion bodies before considering switching to more laborious and expensive purification methods.
Diplonema papillatum RtcB-type RNA ligases Classification of RtcB-type RNA ligases in silico modeling Protein purification