Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Master of Science

Program

Biochemistry

Supervisor

Ilka Heinemann

Abstract

The terminal nucleotidyltransferases TENT3A and TENT3B (TENT3A/B) regulate microRNA (miRNA) and messenger RNA (mRNA) stability by 3' end uridylation, leading to degradation by the U-specific exonuclease DIS3L2. Uridylation dependent decay is thought to be a highly specific process, and the prevalence and impact of this pathway on the transcriptome is unexplored. I investigate the prevalence of uridylation dependent decay by transcriptionally profiling HEK 293T cells lacking TENT3A/B. I found while TENT3A/B target a variety of miRNAs, the let-7 family of miRNAs were the most impacted (increased) in TENT3A/B deleted cells. Consequently, let-7 mRNA targets are decreased in abundance in TENT3A/B deleted cells. mRNAs with increased abundance in the deletion strain may be direct targets or TENT3A/B, with transcripts coding for proteins involved in RNA binding, rRNA processing, etc. Finally, the proliferation and adhesion ability of the deletion cells were reduced, indicating the potential of TENT3A/B as cancer treatment targets.

Summary for Lay Audience

DNA is often referred to as the blueprint of life, as it contains all the information needed to build cells and even organs and whole organisms. This genetic information generally flows from DNA to RNA to protein. During the stage after this information flows from DNA to RNA, there are multiple regulatory pathways that tightly control the amount of RNA (abundance). Two enzymes, TENT3A/B, are found to be responsible to help degrade the RNA, reducing their abundance in the cells. In this study, I used the cells that have been engineered to loose these two important enzymes, and extracted the RNA. I analyzed the difference of the RNA abundance between the un-modified cells and the engineered ones. I found 10%-25% of specific types of RNAs are significantly changed in abundance due to the loss of these two enzymes. These changes were found to be associated to multiple biological processes and molecular functions. In addition, this study also shows the cells lacking the two enzymes grow much slower and are less able to attach to the cell culture plates as compared to the cells that have regular level of TENT3A/B. In this thesis, I discussed the regulatory roles that TENT3A/B play in the cells and concluded that the TENT3A/B dependent RNA degradation is a major regulatory pathway, controlling the steady conditions of RNA levels in the cell. Furthermore, since the disruption of RNAs levels were previously found to be linked to multiple cancers, and the cells lacking TENT3A/B were found to grow much slower, TENT3A/B are considered as potential cancer therapy target, marking their significance in future study.

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