ABSTRACT OF THE DISSERTATION
Mass Spectrometric Study of RNA Epigenetic Modifications
Doctor of Philosophy, Environmental Toxicology Graduate Program
University of California, Riverside, March 2015
Dr. Yinsheng Wang, Chairperson
The rising interest in understanding the functions, regulation and maintenance of the epitranscriptome call for robust and accurate analytical methods for the identification and quantification of post-transcriptional modified nucleosides in RNA. Mass spectrometry has become a very powerful tool for bioanalysis which can elucidate the structure of substrates and provide quantitative measurements. The LC-MS-based analytical method, in combination with genetic manipulation, may facilitate the studies in the area of epitranscriptome. In this thesis, I focus on the development of novel MS-based strategies to identify and quantify post-transcriptional modifications present in total RNA and mRNA isolated from mammalian tissues and cultured human cells. Additionally, by using these analytical methods, I was able to discover new enzymes involved in demethylation of mono-methylated cytosine in RNA both in vitro and in vivo.
In Chapter 2, an LC-MS/MS/MS coupled with the stable isotope-dilution method was developed for the sensitive and accurate quantifications of 5-methylcytidine (m5C), 2'-O-methylcytidine (Cm), N6-methyladenosine (m6A) and 2´-O-methyladenosine (Am) in RNA isolated from mammalian cells and tissues. Our results showed that the distributions of these four methylated nucleosides are tissue-specific. We also found that the levels of m5C, Cm and Am are significantly lower (by 6.5-43 fold) in mRNA than in total RNA isolated from HEK293T cells, whereas the level of m6A was slightly higher (by 1.6 fold) in mRNA than in total RNA.
In Chapter 3, I first demonstrated that Tet enzymes can catalyze the formation of 5-hydroxymethylcytidine (5-hmrC) from m5C in vitro. Subsequently, I established a sensitive and accurate LC-MS/MS/MS with the isotope-dilution method to measure the level of 5-hmrC in vivo and further demonstrated that the catalytic domains of all three Tet enzymes as well as full-length Tet3 could induce the formation of 5-hmrC in human cells.
In Chapter 4, I selected four Fe(Π)- and 2-oxoglutarate-dependent dioxygenases: FTO, ALKBH5, ALKBH2, and ALKBH3, to test their demethylase activity towards m5C in RNA in human cells by using the analytical methods established in Chapter 2 & 3. Our results showed that, the level of 5-hmrC is siginificantly decrease wherea the level of m5C is significantly decreased in Alkbh3-/- cells. Our results suggested that ALKBH3 was involved in the demethylation of m5C in RNA.