Degree Type

Thesis

Date of Award

2014

Degree Name

Master of Science

Department

Biochemistry, Biophysics and Molecular Biology

Major

Genetics

First Advisor

Gustavo MacIntosh

Abstract

RNases T2 have been shown to be evolutionarily conserved in plants, deutrostomes and protostomes indicating that this ancient enzyme may be playing a conserved housekeeping role in diverse organisms. Indeed, evidence from studies in Arabidopsis, zebrafish and human suggests that RNases T2 are involved in rRNA recycling under normal conditions. In order to further elucidate the function of these enzymes in animals, we characterized RNase X25, the only member of the RNase T2 family present in Drosophila melanogaster. In this study, we have shown that RNase X25 is ubiquitously expressed throughout the life cycle, is a major contributor of ribonuclease activity in Drosophila and is upregulated under conditions of nutritional stress accompanied by a concomitant upregulation of Atg5, an autophagy marker. These findings suggest that RNase X25 has functions similar to those of RNase T2 in other organisms.

In order to further clarify the mechanism of rRNA uptake by lysosomes for degradation by RNase X25 in Drosophila melanogaster, we characterized the lysosomal associated membrane glycoprotein, Lamp1. Although it has been used extensively as a lysosomal marker in Drosophila, the actual function of this protein within the lysosomal membrane remains elusive in flies. LAMP2, an ortholog of Lamp1 in humans and mice has three splice variants with conserved luminal regions but different cytoplasmic and transmembrane domains. DmelLamp1 shows the strongest homology with LAMP2C among the three splice variants. Recently, the C-terminal regions of DmelLamp1 and LAMP2C were shown to have very high affinity for RNA isolated from mouse brain. LAMP2A has been implicated in chaperone-mediated autophagy, and LAMP2B deficiency has been shown to cause Danon disease, characterized by cardiomyopathy and myopathy, in humans. In the current study, we have shown that, just as in the case of RNase X25, LAMP1 expression is upregulated under nutritional stress. We have also characterized a loss of function mutant for Lamp1 in Drosophila melanogaster. Lamp1 depletion leads to accumulation of autophagic vacuoles in fat body tissue as evidenced by Lysotracker-red (LTR) staining and upregulation of Atg8 expression. These results warrant further investigation into the putative role of Lamp1 in rRNA binding and transport to the lumen of lysosomes for degradation by RNase X25 in Drosophila.

Copyright Owner

Ayesha Riaz

Language

en

File Format

application/pdf

File Size

93 pages

Included in

Genetics Commons

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