Understanding the Mystery of R-Loops and Their Relationship to Nucleolar Stress

Genomic instability is an increase in the likelihood of DNA mutations and genetic changes during cell division, a common feature in cancer cells. One cause of genomic instability comes from the aberrant formation of three-stranded nucleic acid structures called R-loops. R-loop form when an RNA strand hybridizes to its complementary strand of DNA, forming a stable RNA:DNA hybrid and displacing one free strand of DNA 1. Accumulation of unscheduled R-loops can lead to replication stress, fork collision, gene expression changes, and double-strand breaks that are underlying causes of genome instability and the development of many diseases, including cancer 2. Many cancers, such as breast, lung, ovarian, and prostate, have all been found to have an accumulation of R-loops 3. The nucleolus is a membrane-less organelle inside the nucleus. It is where ribosomal DNA (rDNA) transcription, ribosomal RNA (rRNA) modification, and assembly of ribosomes occur 4. This is the most energetically intensive process in the cell 4,5. In cancer cells, nucleoli are typically increased in size and/or number due to the increased protein production a cancer cell needs to survive, a sign of poor prognosis in many cancer types 6,7. The nucleolus is also a sensor of cellular or genomic stress and can induce p53 activation upon stress 8. Due to the high volume of transcription occurring in the nucleolus, it is likely that a well-regulated R-loop homeostasis is important to the proper functioning of the nucleolus. While it is well known that the nucleolus responds to a vast array of stressors, it is currently unknown if an increase in genomic R-loops can cause nucleolar stress. I hypothesize that increasing R-loop abundance in cells will cause nucleolar stress. In addition, increasing R-loop abundance will sensitize cancer cells to DNA damage inducing or DNA repair inhibiting chemotherapeutic agents. I tested this hypothesis with the following aims. Aim 1. Determine whether DNA damaging or DNA repair inhibiting chemotherapeutic agents affect R-loop abundance and localization in the nucleus I hypothesized that the addition of DNA damage inducing or DNA repair inhibiting chemotherapeutic agents would alter R-loop abundance and localization in cells. My results show that selected DNA damage inducing or DNA repair inhibiting chemotherapeutic agents including Cisplatin and Olaparib slightly increase the abundance of R-loops in cells, but none of the tested chemotherapeutic agents alter the relative location of R-loops in the nucleus. Aim 2. Determine whether R-loops are concentrated in the nucleolus and whether increasing the abundance of R-loops can activate the nucleolar stress pathway I hypothesized that R-loops were concentrated in the nucleolus, and increasing R-loops would activate the nucleolar stress pathway. My immunostaining results show that R-loops appear to be concentrated in the nucleolus and increasing R-loop abundance in cells partially activates the nucleolar stress pathway. Aim 3. Determine whether increasing R-loop levels decreases cell viability I hypothesized that increasing R-loop levels with an addition of DNA damaging, and DNA repair inhibiting drugs and knockdown of Senataxin would activate the nucleolar stress pathway leading to a decrease in cell viability. Unfortunately, no reliable data could be gathered from this aim. The results in this aim were unreliable due to an inconsistent cell counting method and low cell seeding density.