Investigating the stress sensitivity of human RNA ligase Rlig1 deficient cells via proteomics and high-throughput screening

Abstract

Maintenance and repair of polynucleotides are essential processes across all life forms. While DNA repair is well-researched, RNA repair remains largely unexplored. The recent discovery of Rlig1, a 5‘-3’ RNA ligase, could represent a significant step toward the elucidation of further RNA maintenance mechanisms. This thesis employed various approaches to investigate the functional role of Rlig1 in human cells. In the first chapter of this thesis, confocal microscopy of overexpressed Rlig1 revealed a cytosolic localisation with occasional aggregation. CRISPR/Cas9-generated Rlig1-deficient HEK293 cells displayed no phenotypic differences under physiological conditions but showed heightened sensitivity to oxidative stress induced by menadione. This sensitivity was demonstrated by increased cell death and reduced RNA integrity, particularly of the 28S rRNA. As ROS levels generated by menadione treatment were comparable between the knock-out and the parental cell line, Rlig1 appears to be directly involved in RNA-based stress response. However, attempts to rescue Rlig1-KO cells by retroactive overexpression of Rlig1 were unsuccessful, suggesting additional cellular roles for Rlig1 beyond its enzymatic activity. In the second part of this work, protein-protein interaction partners of Rlig1 were identified by an affinity enrichment with LC-MS analysis. Enriched protein groups included ribosomal proteins, exosome components, RNA helicases, and DNA base excision repair proteins. Furthermore, Angel2 was enriched and was shown in combination with Rlig1 to repair RNA strands that would result from common RNA strand cleavage events. In further experiments, Rlig1 was proven to spatially interact with the ribosome. While no specific binding pocket of Rlig1 was identified on the ribosome, the RNA ligase seemed to localise preferentially at the large ribosomal subunit, close to rRNA hotspots on the ribosomal surface. Closer examination of the identified DNA base excision repair enzymes revealed that most of the proteins were already known to interact with RNA. The sole exception was the DNA polymerase POLB, which revealed to be able to elongate RNA in subsequent experiments. For this, exclusively nucleotides and no deoxynucleotides were utilised, proving a deliberate extension of RNA by POLB. In the final chapter of this thesis, a semi-automated high-throughput screening identified ten biologically active small molecules that exhibit synthetic lethality with the knock-out of Rlig1, many of which inhibit NF-κB and/or STAT3, generate ROS, or arrest the cell cycle. In conclusion, the findings of this thesis provide a basis for future research into Rlig1’s role in RNA maintenance and repair, supporting the hypothesis of previously unrecognised RNA repair pathways.