Innate immunity and inflammation are central to human health. This was stated succinctly by the Nobel Laureate David Baltimore in 2011: ‘Autoimmunity, cancer and metabolic diseases are all secondary to chronic inflammation. This places inflammation at the heart of modern medicine’. The ‘Damage Model’ of Polly Matzinger proposes that innate immunity does not response to pathogens but responds to danger as defined by cellular damage. Therefore different cellular stresses can cause cellular damage, accidently triggering innate immunity which manifests itself as an autoimmune disease.
For many years my group has studied ADARs that convert adenosine to inosine in dsRNA. In 2014 we were the first to demonstrate that inosine in cellular RNA allows the cell to discriminate between self and non-self RNAs. We showed that the Adar1-/-embryo is dead by day E12.5 as it lacks hematopoietic progenitor cells and has elevated interferon levels. However this mutant can be rescued by a mutation in Mavs-/- demonstrating that Adar1 is an essential component of the innate immune pathway.
There are over 140 different types of RNA modification so we propose that RNA modifications other that inosine play a role in the innate immune response. Therefore we want to isolate and identify different classes of RNAs (not tRNAs and rRNAs) which are enriched in RNA modification, investigate their biological functions if their levels change in autoimmune disorders.
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Name and position
|Mary O'Connell, Ph.D.
|+420 54949 5460|
|Liam Keegan, Ph.D.
|+420 54949 5460|
||+420 54949 5651|
|Dragana Vukić, M.Sc.
||+420 54949 5651|
|Anzer Khan, M.Sc.
||+420 54949 5651|
|Nagraj Sambrani, PhD
||+420 54949 8505|
||+420 54949 4197|
||+420 54949 8505|
|Dagmara Marta Wiatrek-Moumoulidis
||+420 54949 4259|
Supervisor: Mary O´Connell, Ph.D., MS
Recently there has been a surge of interest in RNA modification; the ‘epitranscriptome’, has been established as a major new field. The extent, diversity and consequence of RNA modifications in the transcriptome are unknown. The modifications studied so far can influence translation, folding, binding of proteins and degradation of RNA. The diseases that they have been implicated in range from cancer to neurodegeneration and even obesity. It is impossible to easily discern RNA modifications as RNA Seq use reverse transcriptase that fails to distinguish between canonical and modified bases. Consequently, most researchers focus on immune-precipitations or chemical reactions targeting just one type of modification. In contrast, we use liquid chromatography-mass spectrometry (LC-MS), the gold standard to detect base modifications. This PhD project is to pioneer the use of LC-MS to investigate other modifications. Poly(A) RNA and miRNA samples will be analyzed to detect inosine, m6A, m1A, pseudouridine, and m5C which are the most abundant modifications. Cell culture will be used to examine various parameters such as stress, induction of the immune response and cell aging. The goal will be to elucidate if there is an orchestrated response in RNA modification to a changing environment.
Supervisor: Liam Keegan, Ph.D., MS
Consultant: Mary O'Connell, Ph.D., MS
RNA modification is a new field that is now being referred to as Epitranscriptomics (Keegan et al., 2004; O’Connell et al., 2015). One of my reviews is the basis of this PhD project (McLaughlin and Keegan, 2014). The proposal is to look at protein structure and evolution of ribonucleotide reductases and thymidylate synthetase, the enzymes that now make DNA precursors, to see whether they once introduced enzymatic modifications into dsRNA genomes directly. Currently these are only known to work on RNA mononucleotides. My hypothesis is that they evolved to work on bases within dsRNA genomes first and changed to act on free mononucleotides as genomes came to be based on DNA. This may have been the origin of DNA genomes. Thymidylate synthetase is already known to bind its own RNA and the project will aim to determine if thymidylate synthetase methylates a specific uracil in its own mRNA. The experiment involving ribonucleotide reductase will be to determine if it can work on the most 3’ bases in a short single-stranded RNA or on an internal RNA base. It is possible that some ribonucleotide reductase enzymes, perhaps from some ancient type of cell, may have conserved a greater ability to interact with RNA than the enzymes present in humans and other model organisms. As one of the three classes of ribonucleotide reductases appears to have the potential to bind RNA, this needs to be evaluated by looking for conservation of positive charges on a long channel on the surface of the protein at the active site using sequence alignments, PyMOL and other modelling programs. Then the selected enzymes can be expressed for in vitro tests of RNA interaction and modification. References Keegan, L.P., Leroy, A., Sproul, D., and O'Connell, M.A. (2004). Adenosine deaminases acting on RNA (ADARs): RNA-editing enzymes. Genome Biol 5, 209. McLaughlin, P.J., and Keegan, L.P. (2014). Conflict RNA modification, host parasite co-evolution, and the origins of DNA and DNA-binding proteins1. Biochem Soc Trans 42, 1159-1167. O’Connell, M.A., Mannion, N.M., and Keegan, L.P. (2015). The Epitranscriptome and Innate Immunity. PLoS Genet 11, e1005687.
Supervisor: Mary O'Connell, Ph.D.
One of the major questions in innate immunity is how does a cell distinguish between ‘self’ and ‘non-self’ RNA. Making this distinction is difficult considering that the cell contains many different types of RNA composed of the same 4 bases. However discriminating between self and non-self is crucial as if the cellular response is too slow then the pathogen wins, if it is too fast then there is an autoimmune response. To help distinguish endogenous from pathogenic RNA, mRNAs are capped at their 5’end. Research from our group has also implicated inosine, which is generated by the deamination of adenosine as being an import mark of ‘self’ on the RNA 1. Mutations in the enzymes responsible for this modification; ADARs induce an interferon response which causes Aicardi-Goutieres syndrome in humans 2 and is embryonic lethal in mice. The aim of this project is to determine where in the innate immune pathway do the ADAR proteins act. Also to determine if lacking other RNA modifications can trigger the innate immune response. The project will entail a mixture of biochemistry as well as molecular biology techniques. 1 Mannion, N. M. et al. The RNA-editing enzyme ADAR1 controls innate immune responses to RNA. Cell Rep 9, 1482-1494, doi:10.1016/j.celrep.2014.10.041 (2014). 2 Rice, G. I. et al. Mutations in ADAR1 cause Aicardi-Goutieres syndrome associated with a type I interferon signature. Nature genetics 44, 1243-1248 (2012).
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