MicroRNA regulation of CTP synthase and cytoophidium in Drosophila melanogaster

Abstract

CTPsyn is a crucial metabolic enzyme which synthesizes CTP molecules through the de novo or salvage pathway. It has the extraordinary ability to compartmentalize into filaments termed cytoophidia. Although this property is retained amongst orthologues, and cytoophidia are therefore found across kingdoms, the mechanisms behind their formation remain unknown. Micro-RNAs (miRNAs) are single-stranded RNA with length of 20 - 22 nucleotides, capable of exerting mRNA silencing and degradation as a form of regulation. D. melanogaster itself has a high total gene count to miRNA gene number ratio, alluding to the possibility that CTPsyn may too come under the regulatory effects of these small RNAs. A thorough miRNA overexpression involving 123 UAS-miRNA lines, followed by staining of ovarian cytoophidia dme-egg chambers, revealed a small group of candidates which confer either a lengthening or truncating effect on the structure. Prime candidates are identified on the basis of consistency. MiR-975 and miR-1014 are both cytoophidia-elongating, whereas miR-190 and miR-932 are cytoophidia-shortening. Though target prediction shows that miR-975 and miR-932 do indeed have binding sites on CTPsyn mRNA, in vitro assays instead revealed that none of the four candidates may actually do so. This suggests that the effects asserted by overexpressed miRNAs indirectly reach CTPsyn and its cytoophidia through the actions of middling elements. In silico target prediction and qPCR quantification indicated that, at least for miR-932 and miR-1014, these undetermined elements may be players in fat metabolism. This is the first study to thoroughly investigate miRNAs in connection to CTPsyn expression and activity in any species. The findings presented could serve as a basis for further queries into not only the fundamental aspects of the enzymes regulation, but may uncover new facets of closely related pathways as well.