GSBS Dissertations and Theses

Approval Date

3-11-2010

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Interdisciplinary Graduate Program

Subjects

Drosophila; Drosophila Proteins; RNA, Untranslated; RNA, Small Interfering; MicroRNAs; Dissertations, UMMS

Abstract

Since the discovery in 1993 of the first small silencing RNA, a dizzying number of small RNAs have been identified, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). These classes differ in their biogenesis, modes of target regulation and in the biological pathways they regulate.

Historically, siRNAs were believed to arise only from exogenous double-stranded RNA triggers in organisms lacking RNA-dependent RNA polymerases. However, the discovery of endogenous siRNAs in flies expanded the biological significance of siRNAs beyond viral defense. By high throughput sequencing we identified Drosophila endosiRNAs as 21 nt small RNAs, bearing a 2´-O-methyl group at their 3´ ends, and depleted in dicer-2 mutants.

Methylation of small RNAs at the 3´ end in the soma, is a consequence of assembly into a mature Argonaute2-RNA induced silencing complex. In addition to endo-siRNAs, we observed certain miRNAs or their miRNA* partners loading into Argonaute2. We discovered, that irrespective of its biogenesis, a miRNA duplex can load into either Argonaute (Ago1 or Ago2), contingent on its structural and sequence features, followed by assignment of one of the strands in the duplex as the functional or guide strand. Usually the miRNA strand is selected as the guide in complex with Ago1 and miRNA* strand with Ago2.

In our efforts towards finding 3´ modified small RNAs in the fly soma, we also discovered 24-28nt small RNAs in certain fly genotypes, particularly ago2 and dcr-2 mutants. 24-28nt small RNAs share many features with piRNAs present in the germline, and a significant fraction of the 24-28nt small RNAs originate from similar transposon clusters as somatic endo-siRNAs. Therefore the same RNA can potentially act as a precursor for both endo-siRNA and piRNA-like small RNA biogenesis. We are analyzing the genomic regions that spawn somatic small RNAs in order to understand the triggers for their production. Ultimately, we want to attain insight into the underlying complexity that interconnects these small RNA pathways.

Dysregulation of small RNAs leads to defects in germline development, organogenesis, cell growth and differentiation. This thesis research provides vital insight into the network of interactions that fine-tune the small RNA pathways. Understanding the flow of information between the small RNA pathways, a great deal of which has been revealed only in the recent years, will help us comprehend how the pathways compete and collaborate with each other, enabling each other’s optimum function.

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