Date of Award

1-1-2017

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Nanoscale Science and Engineering

Program

Nanoscale Engineering

Content Description

1 online resource (ii, xv, 132 pages) : illustrations (some color)

Dissertation/Thesis Chair

Thomas J Begley

Committee Members

Nathaniel Cady, Xinxin Ding, Gabriele Fuchs, Andres J Melendez, Srivathsan Ranganathan

Keywords

ALKBH8, Colorectal Caner, CRISPR/Cas9, Graphene Nanopore, TRM9L, tRNA Modifications, Transfer RNA, Colon (Anatomy), Rectum, Nanopores

Subject Categories

Biomedical Engineering and Bioengineering | Genetics | Nanoscience and Nanotechnology

Abstract

tRNA modifications can be considered epitranscriptomic signaling components that regulate translation and play integral roles in stress response pathways. As such, tRNA modification enzymes have roles in cancer etiology and potential utility as biomarkers of pathological states. For my thesis project I have used computational and wet bench approaches to study tRNA modification systems. Chapter two of my thesis deals with tRNA modification detection, as current methods are costly, time consuming, and require RNA fragmentation. I present a single-molecule-based approach for RNA modification detection, which involves in slico studies using a 5-layered graphene nanopore. Our simulations using a 1.5 nm nanopore demonstrated that there was a 30% increase in the resistance of the pore when the localized uridine (U) was modified to 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um) while the other U-based modifications showed a modest yet significant increase in their respective nanopore resistances. We have compared the results between nanopores of various sizes to support the idea that a 1.5 nm graphene nanopore can be used to differentially detect modified U. In addition, our computational studies will aid in the design, optimization, and fabrication of graphene nanopore devices for tRNA modification detection. In chapter 3 of my thesis I have developed novel reagents to gene edit the tRNA wobble U methyltransferase alkylation repair homolog 8 (ALKBH8). Notably ALKBH8 is required for 5-methoxycarbonylmethyluridine (mcm5U) and mcm5Um tRNA modifications, which are required for the optimal translation of selenoproteins by stop-codon recoding. Selenoproteins are crucial to regulating reactive oxygen species (ROS) levels, which is important during stress responses and adapting to tumor microenvironments.1-3 I present my work using the CRISPR/Cas9 gene editing technique to target ALKBH8 in colorectal cancer models, to eventually assess the effect of the loss of ALKBH8 function has on the cell lines. I have developed a CRISPR construct that targets the N-terminal of ALKBH8, and produced stable ALKBH8-CRISPR-edited SW620 cell line, a highly metastatic colorectal cancer model. In the fourth chapter of my thesis I have studied the ALKBH8 homolog and tumor growth suppressor tRNA methyltransferase 9 like protein (TRM9L). Molecular signals are turned-off or -on in cancers to promote tumor growth and progression. We have observed that TRM9L is turned off in late stage colorectal cancers and that re-expression of TRM9L prevents tumor growth in xenograft models. Despite the homology to the well-studied ALKBH8, little is known about the exact biological function of TRM9L. We approached the study of TRM9L by probing our colorectal cancer model with selenium, based on studies linking low levels of dietary selenium to cancer onset and progression. I present new major findings that TRM9L expression promotes a resistance to selenium exposure and a general change in translation, which drives increased levels of selenoproteins, translated from transcripts with Type II SECIS elements in their 3′ untranslated regions.

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