Homeostatic mechanisms regulate the abundance of many small RNA components. We used Drosophila and mammalian systems to demonstrate a conserved homeostatic system in which the status of miRNA biogenesis controls Argonaute protein stability. Clonal analyses of multiple mutants of core Drosophila miRNA factors revealed that stability of the miRNA effector AGO1 is dependent on miRNA biogenesis. Reciprocally, ectopic transcription of miRNAs within in vivo clones induced accumulation of AGO1, as did genetic interference with the ubiquitin-proteasome system. In mammals, we found that the stability of mAgo2 declined in Dicer knockout cells, and was rescued by proteasome blockade or introduction of either Dicer plasmid or Dicer-independent miRNA constructs. Importantly, Dicer-dependent miRNA constructs generated pre-miRNAs that bind Ago2, but did not rescue Ago2 stability. We conclude that Argonaute levels are finely tuned by cellular availability of mature miRNAs and the ubiquitin-proteasome system.

The essential metabolic enzyme CTP synthase (CTPsyn) can be compartmentalised to form an evolutionarily-conserved intracellular structure termed the cytoophidium. Recently, it has been demonstrated that the enzymatic activity of CTPsyn is attenuated by incorporation into cytoophidia in bacteria and yeast cells. Here we demonstrate that CTPsyn is regulated in a similar manner in Drosophila tissues in vivo. We show that cytoophidium formation occurs during nutrient deprivation in cultured cells, as well as in quiescent and starved neuroblasts of the Drosophila larval central nervous system. We also show that cytoophidia formation is reversible during neurogenesis, indicating that filament formation regulates pyrimidine synthesis in a normal developmental context. Furthermore, our global metabolic profiling demonstrates that CTPsyn overexpression does not significantly alter CTPsyn-related enzymatic activity, suggesting that cytoophidium formation facilitates metabolic stabilisation. In addition, we show that overexpression of CTPsyn only results in moderate increase of CTP pool in human stable cell lines. Together, our study provides experimental evidence, and a mathematical model, for the hypothesis that inactive CTPsyn is incorporated into cytoophidia.

SUMMARYThe Drosophila Hox gene Ultrabithorax (Ubx) controls the development of thoracic and abdominal segments, allocating segmentspecific features to different cell lineages. Recent studies have shown that Ubx expression is post-transcriptionally regulated by two microRNAs (miRNAs), miR-iab4 and miR-iab8, acting on target sites located in the 3Ј untranslated regions (UTRs) of Ubx mRNAs. Here, we show that during embryonic development Ubx produces mRNAs with variable 3ЈUTRs in different regions of the embryo. Analysis of the resulting remodelled 3ЈUTRs shows that each species harbours different sets of miRNA target sites, converting each class of Ubx mRNA into a considerably different substrate for miRNA regulation. Furthermore, we show that the distinct developmental distributions of Ubx 3ЈUTRs are established by a mechanism that is independent of miRNA regulation and therefore are not the consequence of miR-iab4/8-mediated RNA degradation acting on those sensitive mRNA species; instead, we propose that this is a hard-wired 3ЈUTR processing system that is able to regulate target mRNA visibility to miRNAs according to developmental context. We show that reporter constructs that include Ubx short and long 3ЈUTR sequences display differential expression within the embryonic central nervous system, and also demonstrate that mRNAs of three other Hox genes suffer similar and synchronous developmental 3ЈUTR processing events during embryogenesis. Our work thus reveals that developmental RNA processing of 3ЈUTR sequences is a general molecular strategy used by a key family of developmental regulators so that their transcripts can display different levels of visibility to miRNA regulation according to developmental cues.

CTP synthase is an essential enzyme that plays a key role in energy metabolism. Several independent studies have demonstrated that CTP synthase can form an evolutionarily conserved subcellular structure termed cytoophidium. In budding yeast, there are two isoforms of CTP synthase and both isoforms localize in cytoophidium. However, little is known about the distribution of CTP synthase isoforms in Drosophila melanogaster. Here, we report that three transcripts generated at the CTP synthase gene locus exhibit different expression profiles, and three isoforms encoded by this gene locus show a distinct subcellular distribution. While isoform A localizes in the nucleus, isoform B distributes diffusely in the cytoplasm, and only isoform C forms the cytoophidium. In the two isoform C-specific mutants, cytoophidia disappear in the germline cells. Although isoform A does not localize to the cytoophidium, a mutation disrupting mostly isoform A expression results in the disassembly of cytoophidia. Overexpression of isoform C can induce the growth of the cytoophidium in a cell-autonomous manner. Ectopic expression of the cytoophidium-forming isoform does not cause any defect in the embryos. In addition, we identify that a small segment at the amino terminus of isoform C is necessary but not sufficient for cytoophidium formation. Finally, we demonstrate that an excess of the synthetase domain of CTP synthase disrupts cytoophidium formation. Thus, the study of multiple isoforms of CTP synthase in Drosophila provides a good opportunity to dissect the biogenesis and function of the cytoophidum in a genetically tractable organism.

Long noncoding RNAs (lncRNAs) play diverse roles in biological processes. Aedes aegypti ( Ae . aegypti ), a blood-sucking mosquito, is the principal vector responsible for replication and transmission of arboviruses including dengue, Zika, and Chikungunya virus. Systematic identification and developmental characterisation of Ae . aegypti lncRNAs are still limited. We performed genome-wide identification of lncRNAs, followed by developmental profiling of lncRNA in Ae . aegypti . We identified a total of 4,689 novel lncRNA transcripts, of which 2,064, 2,076, and 549 were intergenic, intronic, and antisense respectively. Ae . aegypti lncRNAs share many characteristics with other species including low expression, low GC content, short in length, and low conservation. Besides, the expression of Ae . aegypti lncRNAs tend to be correlated with neighbouring and antisense protein-coding genes. A subset of lncRNAs shows evidence of maternal inheritance; hence, suggesting potential role of lncRNAs in early-stage embryos. Additionally, lncRNAs show higher tendency to be expressed in developmental and temporal specific manner. The results from this study provide foundation for future investigation on the function of Ae . aegypti lncRNAs.

MicroRNA (miRNA) target recognition is largely dictated by short ‘seed’ sequences, and single miRNAs therefore have the potential to regulate a large number of genes. Understanding the contribution of specific miRNA–target interactions to the regulation of biological processes in vivo remains challenging. Here we use transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technologies to interrogate the functional relevance of predicted miRNA response elements (MREs) to post-transcriptional silencing in zebrafish and Drosophila. We also demonstrate an effective strategy that uses CRISPR-mediated homology-directed repair with short oligonucleotide donors for the assessment of MRE activity in human cells. These methods facilitate analysis of the direct phenotypic consequences resulting from blocking specific miRNA–MRE interactions at any point during development.

The mosquito Aedes aegypti (Ae. aegypti) is the most notorious vector of illness-causing viruses such as Dengue, Chikugunya, and Zika. Although numerous genetic expression studies utilizing quantitative real-time PCR (qPCR) have been conducted with regards to Ae. aegypti, a panel of genes to be used suitably as references for the purpose of expression-level normalization within this epidemiologically important insect is presently lacking. Here, the usability of seven widely-utilized reference genes i.e. actin (ACT), eukaryotic elongation factor 1 alpha (eEF1α), alpha tubulin (α-tubulin), ribosomal proteins L8, L32 and S17 (RPL8, RPL32 and RPS17), and glyceraldeyde 3-phosphate dehydrogenase (GAPDH) were investigated. Expression patterns of the reference genes were observed in sixteen pre-determined developmental stages and in cell culture. Gene stability was inferred from qPCR data through three freely available algorithms i.e. BestKeeper, geNorm, and NormFinder. The consensus rankings generated from stability values provided by these programs suggest a combination of at least two genes for normalization. ACT and RPS17 are the most dependably expressed reference genes and therefore, we propose an ACT/RPS17 combination for normalization in all Ae. aegypti derived samples. GAPDH performed least desirably, and is thus not a recommended reference gene. This study emphasizes the importance of validating reference genes in Ae. aegypti for qPCR based research.

Argonaute 1 (Ago1) is a member of the Argonaute/PIWI protein family involved in small RNA-mediated gene regulation. In Drosophila, Ago1 plays a specific role in microRNA (miRNA) biogenesis and function. Previous studies have demonstrated that Ago1 regulates the fate of germline stem cells. However, the function of Ago1 in other aspects of oogenesis is still elusive. Here we report the function of Ago1 in developing egg chambers. We find that Ago1 protein is enriched in the oocytes and also highly expressed in the cytoplasm of follicle cells. Clonal analysis of multiple ago1 mutant alleles shows that many mutant egg chambers contain only 8 nurse cells without an oocyte which is phenocopied in dicer-1, pasha and drosha mutants. Our results suggest that Ago1 and its miRNA biogenesis partners play a role in oocyte determination and germline cell division in Drosophila.