Phylogenetic Analyses and Characterization of RNase X25 from Drosophila melanogaster Suggest a Conserved Housekeeping Role and Additional Functions for RNase T2 Enzymes in Protostomes

Abstract

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.

Emerging connections between RNA and autophagy

“…In line with the presumption that RNA-mediated degradation is a conserved mechanism with importance also in higher organisms, interesting roles for the T2 family of RNases in animals have also been found. 45,49,50 Drosophila melanogaster has a single RNase T2, called RNaseX25, which is elevated in response to nutritional stress and correlates in expression with autophagy markers, supporting the idea that RNA recycling via autophagy also occurs in the fly. 49 In addition, a role for the ortholog of yeast Rny1, Rnaset2 has recently been described in zebrafish.…”

RNASET2 is required for ROS propagation during oxidative stress-mediated cell death

“…34 Drosophila expresses one RNASET2 homolog, RNase X25, which is induced by ROS and promotes cell death when overexpressed. 28,35 Constitutive, whole body knockdown of RNase X25 did not result in viable progeny, underscoring the importance of RNase X25 in general housekeeping functions. However, flies with fat body-specific knockdown of RNase X25 were viable and had similar body weight to WT flies (Figure 7a).…”

“…27 In mammalian cells, expression of RNASET2 family members is induced in response to exposure to hydrogen peroxide, ultraviolet irradiation, and inflammatory stimuli. 28,35 Moreover, overexpression of this enzyme in melanocytes and keratinocytes sensitizes cells to oxidative-stress-induced apoptosis. 28 Our study extends these findings in several important ways.…”

Evidence for autophagy-dependent pathways of rRNA turnover inArabidopsis

“…20,[58][59][60][61] RNS2 is one of 5 RNase T2 enzymes found in Arabidopsis and is the only class II RNase T2 constitutively expressed throughout the plant and throughout development. 60,62 RNase T2 proteins are targeted to organelles of the secretory pathway, including the endoplasmic reticulum, Golgi apparatus, vacuole, lysosome, and associated vesicles, or secreted from the cell.…”