A systems-level analysis of dynamic reprogramming of RNA modifications in the translational control of cellular responses

In addition to the four canonical ribonucleosides (adenosine, uridine, guanosine, cytosine), transfer RNAs (tRNA) and ribosomal RNAs (rRNA) are comprised of more than 100 enzymecatalyzed modifications, with about 20-35 found in any one organism. Many of these modifications are highly conserved in all domains of life, which suggests important biological roles for RNA modifications in cell physiology. Several recent studies have demonstrated that individual tRNA modifications and their biosynthetic pathways affect cellular stress responses. The presence of 20-35 different RNA modifications in all translationally-related non-coding RNAs suggested the possibility of systems behavior of RNA modifications in translational facets of cellular responses. The studies presented in this thesis utilize a quantitative systems-level approach to test the hypothesis that the spectrum of tRNA modifications represents a cellular program involved in modulating stress response pathways. To initiate these studies, a novel mass spectrometric platform was developed to characterize and quantify the spectrum of modified ribonucleosides in an organism, starting with the ~25 ribonucleosides in S. cerevisiae tRNA. This approach was used to compare tRNA modification spectra from cells exposed to four mechanistically distinct toxicants: hydrogen peroxide, methyl methanesulfonate, arsenite, and hypochlorite. Multivariate statistical analysis revealed both doseand agent-specific signatures in the relative quantities of tRNA modifications. Further, modifications that change significantly after exposure were shown to confer resistance to the cytotoxicity of the agent. These observations demonstrate the dynamic nature of tRNA modifications and their critical role in translational control of cellular stress responses. Also, application of the mass spectrometric method revealed several new biosynthetic pathways for tRNA modifications in yeast. These studies comprise Chapters 2 and 3. Chapter 4 is aimed at characterizing the link between tRNA modifications and translational control of cellular responses. One of the tRNA modifications that increased significantly following exposure of yeast to hydrogen peroxide is 5-methylcytosine (m5 C), which is located at the wobble position of the leucine tRNA for coding UUG. This suggested that it might affect translation of mRNA containing this codon. While there are 6 codons for leucine, the usage of the codon UUG for specifying leucine in the set of homologous ribosomal proteins differs widely. Using proteomics approach, it was demonstrated that m5C regulates the levels of the homologous ribosomal protein genes rp/22a and rp/22b, with hydrogen peroxide exposure causing an increase in the proportion of ribosomes containing rpI22a. Further, loss of rp/22a conferred sensitivity to hydrogen peroxide exposure. These results suggest that the system of tRNA modifications controls cellular responses partly by determining the composition of ribosomes involved in the selective translation of critical response proteins. As observed in Chapter 3, tRNA modifications spectrum changes specifically in responses to mechanistically distinct toxic agents; in Chapter 5, a series of studies was designed to test the hypothesis that each of these unique signatures represents a common response to different toxicant classes. To test this hypothesis, yeast cells were exposed to four different oxidizing agents (hydrogen peroxide, tert-butyl hydroperoxide, peroxynitrite, and gamma-radiation) and five different alkylating agents (methyl methanesulfonate, ethyl methanesulfonate, isopropyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, and N-nitroso-N-methylurea) at concentrations producing similar levels of cytotoxicity. The spectrum of tRNA modifications was then quantified and the results subjected to multivariate statistical analysis to identify consistent patterns. The results reveal class-specific patterns of changes, with distinct tRNA modification spectra for oxidants and alkylating agents. At a finer level of analysis, the studies revealed subclass signatures for SN1 and SN2 alkylating agents. The results from these experiments were used to develop a data-driven model that predicts exposures to the two classes of toxic agents accurately. Such a model may be useful for assessing ribonucleoside spectra as biomarkers of exposure. Appendix A describes the preliminary characterization of the spectrum of modified ribonucleosides from Mycobacterium bovis BCG tRNA. Surveys of tRNA enzymatic hydrolysates with mass spectrometric techniques reveal the presence of modified ribonucleosides that are highly conserved among various species of organisms, as well as candidates of novel modifications. Advisor: Peter C. Dedon Title: Underwood-Prescott Professor of Toxicology and Biological Engineering Singapore Research Professor of Infectious Disease Deputy Director of MIT Center for Environmental Health Sciences Faculty Member of Chulabhorn Graduate Research Institute Dedicated to my parents Kwok Ping Chan and So Ngan Woo For their counsel and support in all these years And to myfiancie Man Yin Leung Who keeps me full with love every day