Functional analysis of the nuage, a unique germline organelle, in Drosophila melanogaster

Abstract

Nuage is an electron-dense perinuclear structure that is known to be a hallmark of the animal germline cells. Although the conservation of the nuage throughout evolution accentuates its essentiality, its role(s) and the exact mechanism(s) by which it functions in the germline still remain unknown. In this thesis, I report a novel nuage component Krimper (KRIMP) in Drosophila melanogaster, and show that it ensures the repression of retroelements in the female germline. krimp loss-of function allele exhibits female sterility, defects in karyosome formation and oocyte polarity, and precocious oskar translation. These phenotypes are commonly observed in two other nuage component mutants spindle-E and aubergine, which are also known to mediate RNA interference. This therefore suggests a shared underlying defect that utilises RNA silencing. In the nuage component mutants, retroelements HeT-A, TART, I-element, and mst40 are derepressed to different extents. De-repression of retroelements appears to correlate with the down-regulation of a unique class of small RNAs, termed as P-element-induced wimpy testes (Piwi)-interacting RNAs (piRNAs). This therefore suggests that the nuage functions as a specialised ¿centre¿ to govern genome fidelity in the germline cells via piRNA-mediated gene regulation. Besides localising to the perinuclear sites, the nuage/piRNA pathway components are found in cytoplasmic foci that also contain retroelement transcripts, anti-sense piRNAs, and proteins involved in mRNA degradation. These mRNA degradation proteins Decapping Proteins, Maternal Expression at 31B (Me31B, a decapping activator), and II Pacman, are normally thought of as components of processing bodies. In spindle-E and aubergine mutants that lack piRNA production, piRNA pathway proteins no longer overlap mRNA degradation proteins. Concomitantly, spindle-E and aubergine mutant ovaries show an accumulation of full-length retroelement transcripts and prolonged stabilisation of HeT-A mRNA, supporting the role of piRNAs in mediating posttranscriptional retroelement silencing. HeT-A mRNA is de-repressed in mRNA degradation mutants, indicating that these enzymes also aid in removing full-length transcripts and/or decay intermediates.