Reduced genome stability and DNA damage are now well-established as drivers of multimorbidity, leading to a number of age associated disorders such as cancer, dementia and inflammatory disease. Ribonucleotide misincorporation in DNA is by far the most abundant source of genomic damage and the ability to model in vertebrates represents an important advance to study common mechanisms underpinning multiple pathologies, moving away from the single disease model. The RNaseH2 enzyme complex fulfils essential functions in high-fidelity removal and repair of misincoporated ribonucleotides. These ribonucleotides cause distortion in the DNA and are more reactive leading to DNA breaks, thereby threatening the integrity of the genome. In addition, RNaseH2 is essential for resolution of RNA/DNA hybrids (R-loops) that occur naturally, or as a result of certain genetic diseases that involve repeat expansions in the DNA, like amyotropic lateral sclerosis (ALS). These two functions are not only required in the nucleus but also in the mitochondria, but here RNase2 is not present, and there may be a currently unidentified protein that fulfils this function in these organelles.
We have created a model in the zebrafish that lacks RNaseH2a function, the essential catalytic subunit of RNaseH2, and found, very surprisingly, that these fish are viable, which provides a unique opportunity to study aging, and genome stability under circumstances where ribonucleotides and R-loops are not properly processed. One highly surprising phenotype that was discovered, is that homozygous males and females can only produce progeny that are highly abnormal, show neurodegeneration and die within a few days of development.
Aim and Objectives
We hypothesize that RNaseH2a mutant animals will show a premature aging and inflammatory phenotypes, as a result of defects in nuclear genome maintenance. We hypothesise that although the fish may be viable the presence of R-loops or ribonucleotides may specifically accumulate in the germline, accelerating ageing of this tissue in particular.
The student will do a detailed molecular characterisation of this mutant in order to explain and understand the observed phenotype, using specific mutant forms of RNAseH2a the student will rest whether the embryonic phenotypes are mainly due to R-loops or ribonucleotides. We are also aiming to examine the consequence of RNAseH2a deficiency on senescence using the p21 reporter line developed at HELSI, and compare and combine this with a Top1MT mutant which should be defective in removal of ribonucleotides specifically in mitochondrial DNA.
Methodology
The student involved will study the precise phenotype or the RNaseH2a mutants, using morphological (eg. cachexia), histology (eg. analysis of gonadal morphology) and molecular markers of aging (eg. p21, Senescence-Associated beta-Galactosidase) and inflammation. This will show if there are aging phenotypes observed and determine if aging occurs generally or segmentally.
Differences between levels of ribonucleotides/R-loops various tissues will be assessed using established techniques in the labs of the supervisors, and we aim to compare germline vs somatic DNA in these mutants. We will analyse changes in the levels of these over time and in different tissues using for instance Comet assays, but also plan to use NGS/Mass Spec methods, to localise and quantify ribonucleotide levels in the genome.
We will microinject in vitro transcribed RNAs encoding various mutant forms of RNAseH2 to test whether R-loop resolution or ribonucleotide removal is the main cause of the embryonic phenotype.
Science Graduate School
As a PhD student in one of the science departments at the University of Sheffield, you’ll be part of the Science Graduate School. You’ll get access to training opportunities designed to support your career development by helping you gain professional skills that are essential in all areas of science. You’ll be able to learn how to recognise good research and research behaviour, improve your communication abilities and experience the breadth of technologies that are used in academia, industry and many related careers. Visit www.sheffield.ac.uk/sgs to learn more.
Continue with Facebook
