Characterization of Nrl1 protein in S. Pombe: a link between RNAi and Pol II?

Recent evidence indicates the existence of a physical and functional link between RNA-interference (RNAi) and RNA polymerase II (Pol II) throughout the eukaryotic kingdom. However, the role of this complex partnership on the metazoan heterochromatin landscape remains still unexplored. In Caenorhabditis elegans, the NRDE-2 protein connects RNAi and Pol II inhibition, in a process of co-transcriptional gene silencing (CTGS). By in silico analysis, I have identified the ortholog of C. elegans NRDE-2 in S. pombe, and I have named it NRde-2 Like protein 1 or Nrl1. In the project I propose here, I want to investigate whether and how Nrl1 may also represent a link between RNAi and Pol II in S. pombe. Moreover, I want to analyse the transcriptional behaviour of Pol II at silenced loci, and whether RNAi factors and/or Nrl1 itself may influence this behaviour. To tackle these questions I will 1) analyse the function of Nrl1 in both wild-type (WT) and Nrl1 deleted (Nrl1 ) strains to asses its physical interaction and functional interplay with the RNAi machinery in heterochromatin gene silencing; 2) measure Pol II occupancy and transcription at heterochromatic loci targeted by RNAi in WT, RNAi mutant and Nrl1 strains. Since Nrl1 has orthologs also in Arabidopsis, Drosophila and human this study will produce important contributions in the field of metazoan RNAi-mediated CTGS, going far beyond the specific yeast system I will use in this work.

In this project we have characterized the evolutionarily conserved protein Nrl1, and uncovered its role in genome stability using the yeast Schizosaccharomyces pombe as model organism. Our DNA is constantly exposed to damage not only from chemicals in our environment but also from the fundamental processes required for translating its information into proteins- such as pre-mRNA processing. While the inability to repair DNA damage efficiently is a known cause of cancer, defects in pre-mRNA processing can also have oncogenic effects- in part via the formation of RNA-DNA hybrids called R-loops. It is likely that failures in DNA repair and R-loop prevention contribute synergistically to DNA damage. Yet direct evidence for this model is still missing. In this study, we provide evidence that Nrl1 associates physically and functionally with pre-mRNA processing factors, and ensures both efficient DNA repair and R-loop suppression. Our results support a model for a novel mechanism of DNA damage, in which R-loops sequester key DNA repair enzymes, such as Rad51, thus inhibiting their repair function. Importantly, human cells also have a protein similar to Nrl1, whose levels are altered in cancer. This study lays the foundation for exploring the role of this protein and other pre-mRNA processing factors in DNA repair and R-loop formation during tumor development.