Date of Award

Fall 2016

Document Type

Dissertation - Restricted

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Anderson, James T.

Second Advisor

Abbott, Allison

Third Advisor

Maki, James

Fourth Advisor

Petrella, Lisa

Fifth Advisor

Schlappi, Michael

Abstract

RNA surveillance via the nuclear exosome requires cofactors such as the helicase SKIV2L2 to process and degrade certain noncoding RNAs. This dissertation aimed to characterize the phenotype associated with RNAi knockdown of SKIV2L2 in two murine cancer cell lines: Neuro2A and P19. Skiv2l2 knockdown in Neuro2A and P19 cells induced changes in gene expression indicative of cell differentiation and reduced cellular proliferation. Analysis of the cell cycle revealed defective progression through mitosis following SKIV2L2 depletion. These results indicate that SKIV2L2 enhances mitotic progression, thereby maintaining cancer cell proliferation and preventing differentiation. Indeed, SKIV2L2 levels were found to be downregulated during chemically induced differentiation, further implicating SKIV2L2 in maintaining proliferation and multipotency in cancer cell lines. Because SKIV2L2 targets RNAs for processing and degradation via the nuclear exosome, it was hypothesized that with SKIV2L2 depletion, the accumulation of some RNA target triggers mitotic arrest. In search of such a target, RNA-seq was utilized to identify RNAs that were elevated in Skiv2l2 knockdown cells. SKIV2L2 depletion resulted in the accumulation of non-coding RNAs, intergenic RNAs, ribosomal protein mRNAs, and replication-dependent histone mRNAs. Given that the regulation of histone mRNAs is tightly linked to the cell cycle, further experiments were conducted to confirm SKIV2L2 targets histone mRNAs for degradation. RNA immunoprecipitation demonstrated direct binding between SKIV2L2 and histone mRNAs, and RNA degradation assays showed that the half-life of histone mRNAs doubles with SKIV2L2 depletion. The resulting histone imbalance following loss of SKIV2L2-directed RNA surveillance could impede mitotic progression, resulting in mitotic defects and indirectly triggering differentiation.

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