Access Type

Open Access Dissertation

Date of Award

January 2013

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

First Advisor

Victoria H. Meller

Abstract

In humans and flies, females have two X chromosomes but males have one X chromosome and one Y chromosome. This leads to a fatal imbalance in X-linked gene expression in one sex. In mammals and in the fruit fly Drosophila, modulation of X chromosome expression is critical for survival. This process is termed dosage compensation. Flies increase expression from the male X chromosome two-fold. This is achieved by the Male Specific Lethal (MSL) complex, which consists of two large, non-coding RNA on the X transcripts (roX1 and roX2) and five proteins. The roX RNAs have a critical role in complex localization to the X chromosome. Simultaneous mutation of roX1 and roX2 reduces X localization of the MSL proteins, lowers X-linked expression and reduces male survival. Using roX1 roX2 mutants, we performed genetic studies to identify modifiers of X chromosome recognition. In spite of a lack of expression in somatic tissues, the Y chromosome is a potent modifier of the roX1 roX2 phenotype. I postulated that the Y chromosome could affect dosage compensation through a small RNA-dependent pathway, and performed a screen of RNAi mutations. This screen identified four siRNA genes that, when mutated, enhance roX1 roX2 male lethality and disrupt MSL localization to the X chromosome. The role of the siRNA pathway in dosage compensation prompted an investigation of potential sources of siRNA. A class of 1.688g/cm3 satellite-related repeats is exclusive to the X chromosome (1.688X). These are transcribed, and thus capable of generating siRNA in animals. Ectopic expression of long single stranded 1.688X RNA reduced roX1 roX2 male survival. In contrast, expression of double stranded 1.688X hairpin RNA produced high levels of corresponding small RNA and dramatically rescued roX1 roX2 male survival. MSL localization to the X chromosome was partially restored in flies expressing 1.688X hairpin RNA. Rescue of roX1 roX2 males was dependent upon the siRNA genes Dcr2 and Ago2. These studies reveal that small RNA from X-linked repeats acts through the siRNA pathway to promote X chromosome recognition. I postulate that the 1.688X RNA repeats underline X chromosome identity. Future studies exploring this process will help us to understand the molecular basis for exclusive modification of the X chromosome.

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