Date

11-6-2013

UMMS Affiliation

Graduate School of Biomedical Sciences, Interdisciplinary Graduate Program

Document Type

Dissertation, Doctoral

Subjects

Dissertations, UMMS; RNA, Small Interfering; RNA Interference; DNA Transposable Elements; Drosophila Proteins

Disciplines

Molecular genetics

Abstract

piRNAs guide PIWI proteins to silence transposons in animal germ cells. In Drosophila, the heterochromatic piRNA clusters transcribe piRNA precursors to be transported into nuage, a perinuclear structure for piRNA production and transposon silencing. At nuage, reciprocal cycles of piRNA-directed RNA cleavage—catalyzed by the PIWI proteins Aubergine (Aub) and Argonaute3 (Ago3) in Drosophila—destroy the sense transposon mRNA and expand the population of antisense piRNAs in response to transposon expression, a process called the Ping-Pong cycle. Heterotypic Ping-Pong between Aub and Ago3 ensures that antisense piRNAs predominate.

My thesis research mainly focuses on two fundamental questions about the piRNA production: How does the germ cell differentiate piRNA precursor from mRNAs for piRNA biogenesis? And what is the mechanism to impose Aub Ping-Pong with Ago3? For the first question, we show that the HP1 homolog protein Rhino marks the piRNA cluster regions in the genome for piRNA biogenesis. Rhino seems to anchor a nuclear complex that suppresses cluster transcript splicing, which may differentiate piRNA precursors from mature mRNAs. Moreover, LacI::Rhino fusion protein binding suppresses splicing of a reporter transgene and is sufficient to trigger de novo piRNA production from a trans combination of sense and antisense transgenes. For the second question, we show that Qin, a new piRNA pathway factor contains both E3 ligase and Tudor domains, colocalizes with Aub and Ago3 in nuage, enforces heterotypic Ping- Pong between Aub and Ago3. Loss of qinleads to less Ago3 binding to Aub, futile Aub:Aub homotypic Ping-Pong prevails, antisense piRNAs decrease, many families of mobile genetic elements are reactivated, DNA damage accumulates in the germ cells and flies are sterile.

Available for download on Thursday, November 20, 2014

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