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Scholarly Works (5 results)

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UC Riverside Electronic Theses and Dissertations (2015)

ABSTRACT OF THE DISSERTATION

Mass Spectrometric Study of RNA Epigenetic Modifications

by

Lijuan Fu

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.

    Cover page: Mass Spectrometric Study of RNA Epigenetic Modifications
    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2014)
    DDB2 exhibits a high affinity toward UV-damaged DNA, and it is involved in the initial steps of global genome nucleotide excision repair. Mutations in the DDB2 gene cause the genetic complementation group E of xeroderma pigmentosum, an autosomal recessive disease manifested clinically by hypersensitivity to sunlight exposure and an increased predisposition to skin cancer. Here we found that, in human cells, the initiating methionine residue in DDB2 was removed and that the N-terminal alanine could be methylated on its α-amino group in human cells, with trimethylation being the major form. We also demonstrated that the α-N-methylation of DDB2 is catalyzed by the N-terminal RCC1 methyltransferase. In addition, a methylation-defective mutant of DDB2 displayed diminished nuclear localization and was recruited at a reduced efficiency to UV-induced cyclobutane pyrimidine dimer foci. Moreover, loss of this methylation conferred compromised ATM (ataxia telangiectasia mutated) activation, decreased efficiency in cyclobutane pyrimidine dimer repair, and elevated sensitivity of cells toward UV light exposure. Our study provides new knowledge about the posttranslational regulation of DDB2 and expands the biological functions of protein α-N-methylation to DNA repair.
      Cover page: α-N-Methylation of Damaged DNA-binding Protein 2 (DDB2) and Its Function in Nucleotide Excision Repair*
      • Article
      • Peer Reviewed
      UC Riverside Previously Published Works (2013)
      Cytosine DNA methylation is an epigenetic mark in most eukaryotic cells that regulates numerous processes, including gene expression and stress responses. We performed a genome-wide analysis of DNA methylation in the human malaria parasite Plasmodium falciparum. We mapped the positions of methylated cytosines and identified a single functional DNA methyltransferase (Plasmodium falciparum DNA methyltransferase; PfDNMT) that may mediate these genomic modifications. These analyses revealed that the malaria genome is asymmetrically methylated and shares common features with undifferentiated plant and mammalian cells. Notably, core promoters are hypomethylated, and transcript levels correlate with intraexonic methylation. Additionally, there are sharp methylation transitions at nucleosome and exon-intron boundaries. These data suggest that DNA methylation could regulate virulence gene expression and transcription elongation. Furthermore, the broad range of action of DNA methylation and the uniqueness of PfDNMT suggest that the methylation pathway is a potential target for antimalarial strategies.
        Cover page: Genome-wide mapping of DNA methylation in the human malaria parasite Plasmodium falciparum.
        • Article
        • Peer Reviewed
        UC Riverside Previously Published Works (2020)
        5-Methylcytosine is found in both DNA and RNA; although its functions in DNA are well established, the exact role of 5-methylcytidine (m5C) in RNA remains poorly defined. Here we identified, by employing a quantitative proteomics method, multiple candidate recognition proteins of m5C in RNA, including several YTH domain-containing family (YTHDF) proteins. We showed that YTHDF2 could bind directly to m5C in RNA, albeit at a lower affinity than that toward N6-methyladenosine (m6A) in RNA, and this binding involves Trp432, a conserved residue located in the hydrophobic pocket of YTHDF2 that is also required for m6A recognition. RNA bisulfite sequencing results revealed that, after CRISPR-Cas9-mediated knockout of the YTHDF2 gene, the majority of m5C sites in rRNA (rRNA) exhibited substantially augmented levels of methylation. Moreover, we found that YTHDF2 is involved in pre-rRNA processing in cells. Together, our data expanded the functions of the YTHDF2 protein in post-transcriptional regulations of RNA and provided novel insights into the functions of m5C in RNA biology.
          Cover page: YTHDF2 Binds to 5‑Methylcytosine in RNA and Modulates the Maturation of Ribosomal RNA
          • Article
          • Peer Reviewed
          UC Riverside Previously Published Works (2013)
          Cytosine DNA methylation is an epigenetic mark in most eukaryotic cells that regulates numerous processes, including gene expression and stress responses. We performed a genome-wide analysis of DNA methylation in the human malaria parasite Plasmodium falciparum. We mapped the positions of methylated cytosines and identified a single functional DNA methyltransferase (Plasmodium falciparum DNA methyltransferase; PfDNMT) that may mediate these genomic modifications. These analyses revealed that the malaria genome is asymmetrically methylated and shares common features with undifferentiated plant and mammalian cells. Notably, core promoters are hypomethylated, and transcript levels correlate with intraexonic methylation. Additionally, there are sharp methylation transitions at nucleosome and exon-intron boundaries. These data suggest that DNA methylation could regulate virulence gene expression and transcription elongation. Furthermore, the broad range of action of DNA methylation and the uniqueness of PfDNMT suggest that the methylation pathway is a potential target for antimalarial strategies.
            Cover page: Genome-wide Mapping of DNA Methylation in the Human Malaria Parasite Plasmodium falciparum
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