Ribonucleases belonging to the RNase T2 family are enzymes associated with the secretory pathway that are almost absolutely conserved in all eukaryotes. Studies in plants and vertebrates suggest they have an important housekeeping function in rRNA recycling. However, little is known about this family of enzymes in protostomes. We characterized RNase X25, the only RNase T2 enzyme in Drosophila melanogaster. We found that RNase X25 is the major contributor of ribonuclease activity in flies as detected by in gel assays, and has an acidic pH preference. Gene expression analyses showed that the RNase X25 transcript is present in all adult tissues and developmental stages. RNase X25 expression is elevated in response to nutritional stresses; consistent with the hypothesis that this enzyme has a housekeeping role in recycling RNA. A correlation between induction of RNase X25 expression and autophagy was observed. Moreover, induction of gene expression was triggered by oxidative stress suggesting that RNase X25 may have additional roles in stress responses. Phylogenetic analyses of this family in protostomes showed that RNase T2 genes have undergone duplication events followed by divergence in several phyla, including the loss of catalytic residues, and suggest that RNase T2 proteins have acquired novel functions. Among those, it is likely that a role in host immunosuppression evolved independently in several groups, including parasitic Platyhelminthes and parasitoid wasps. The presence of only one RNase T2 gene in the D. melanogaster genome, without any other evident secretory RNase activity detected, makes this organism an ideal system to study the cellular functions of RNase T2 proteins associated with RNA recycling and maintenance of cellular homeostasis. On the other hand, the discovery of gene duplications in several protostome genomes also presents interesting new avenues to study additional biological functions of this ancient family of proteins.

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The endolysosomal system not only is an integral part of the cellular catabolic machinery that processes and recycles nutrients for synthesis of biomaterials, but also acts as signaling hub to sense and coordinate the energy state of cells with growth and differentiation. Lysosomal dysfunction adversely influences vesicular transport-dependent macromolecular degradation and thus causes serious problems for human health. In mammalian cells, loss of the lysosome associated membrane proteins LAMP1/2 strongly impacts autophagy and cholesterol trafficking. Here we show that the previously uncharacterized Drosophila Lamp1 is a bona fide homolog of vertebrate LAMP1/2. Surprisingly and in contrast to Lamp1/2 double mutant mice, Drosophila Lamp1 is not required for viability or autophagy, suggesting that autophagy defects in Lamp1/2 mutants may have indirect causes. However, Lamp1 deficiency results in an expansion of the acidic compartment in flies. Furthermore, we find that Lamp1 mutant larvae have defects in lipid metabolism as they show elevated levels of sterols and diacylglycerols (DAGs). Since DAGs are the main lipid species used for transport though the hemolymph (blood) in insects, our results indicate broader functions of Lamp1 in lipid transport. Our findings make Drosophila an ideal model to study the role of LAMP proteins in lipid assimilation without the confounding effects of their storage and without interfering with autophagic processes.

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 Cterminal 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 vi 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. CHAPTER 2 CHARACTERIZATION OF RIBONUCLEASE X25: THE ONLY RNASE T2 ENZYME IN DROSOPHILA MELANOGASTER This chapter is part of the following publication,