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The current toolkit for genetic manipulation in the model animal Drosophila melanogaster is extensive and versatile but not without its limitations. Here, we report a powerful and heritable method to knockdown gene expression in D. melanogaster using the self-cleaving N79 hammerhead ribozyme, a modification of a naturally occurring ribozyme found in the parasite Schistosoma mansoni. A 111-bp ribozyme cassette, consisting of the N79 ribozyme surrounded by insulating spacer sequences, was inserted into 4 independent long noncoding RNA genes as well as the male-specific splice variant of doublesex using scarless CRISPR/Cas9-mediated genome editing. Ribozyme-induced RNA cleavage resulted in robust destruction of 3' fragments typically exceeding 90%. Single molecule RNA fluorescence in situ hybridization results suggest that cleavage and destruction can even occur for nascent transcribing RNAs. Knockdown was highly specific to the targeted RNA, with no adverse effects observed in neighboring genes or the other splice variants. To control for potential effects produced by the simple insertion of 111 nucleotides into genes, we tested multiple catalytically inactive ribozyme variants and found that a variant with scrambled N79 sequence best recapitulated natural RNA levels. Thus, self-cleaving ribozymes offer a novel approach for powerful gene knockdown in Drosophila, with potential applications for the study of noncoding RNAs, nuclear-localized RNAs, and specific splice variants of protein-coding genes., Competing Interests: Conflicts of interest The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The insulin/insulin-like growth factor (IGF) pathway is essential for linking nutritional status to growth and metabolism. MicroRNAs (miRNAs) are short RNAs that are players in the regulation of this process. The miRNA miR-7 shows highly conserved expression in insulin-producing cells across the animal kingdom. However, its conserved functions in regulation of insulin-like peptides (ILPs) remain unknown. Using Drosophila as a model, we demonstrate that miR-7 limits ILP availability by inhibiting its production and secretion. Increasing miR-7 alters body growth and metabolism in an ILP-dependent manner, elevating circulating sugars and total body triglycerides, while decreasing animal growth. These effects are not due to direct targeting of ILP mRNA, but instead arise through alternate targets that affect the function of ILP-producing cells. The Drosophila F-actin capping protein alpha (CPA) is a direct target of miR-7, and knockdown of CPA in insulin-producing cells phenocopies the effects of miR-7 on ILP secretion. This regulation of CPA is conserved in mammals, with the mouse ortholog Capza1 also targeted by miR-7 in β-islet cells. Taken together, these results support a role for miR-7 regulation of an actin capping protein in insulin regulation, and highlight a conserved mechanism of action for an evolutionarily ancient microRNA., (© Endocrine Society 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Genetic variation is prevalent among individuals of the same species and yet the potential effects of genetic variation on developmental outcomes are frequently suppressed. Understanding the mechanisms that are responsible for this suppression is an important goal. Previously, we found that the microRNA miR-9a mitigates the impact of natural genetic variants that promote the development of scutellar bristles in adult Drosophila. Here we find that miR-9a does not affect the impact of genetic variants that inhibit the development of scutellar bristles. We show this using both directional and stabilizing selection in the laboratory. This specificity of action suggests that miR-9a does not interact with all functional classes of developmental genetic variants affecting sensory organ development. We also investigate the impact of miR-9a on a fitness trait, which is adult viability. At elevated physiological temperatures, miR-9a contributes to viability through masking genetic variants that hinder adult viability. We conclude that miR-9a activity in different developmental networks contributes to suppression of natural variants from perturbing development., (Copyright © 2016 by the Genetics Society of America.)

ETS family transcription factors are evolutionarily conserved downstream effectors of Ras/MAPK signaling with critical roles in development and cancer. In Drosophila, the ETS repressor Yan regulates cell proliferation and differentiation in a variety of tissues; however, the mechanisms of Yan-mediated repression are not well understood and only a few direct target genes have been identified. Yan, like its human ortholog TEL1, self-associates through an N-terminal sterile α-motif (SAM), leading to speculation that Yan/TEL1 polymers may spread along chromatin to form large repressive domains. To test this hypothesis, we created a monomeric form of Yan by recombineering a point mutation that blocks SAM-mediated self-association into the yan genomic locus and compared its genome-wide chromatin occupancy profile to that of endogenous wild-type Yan. Consistent with the spreading model predictions, wild-type Yan-bound regions span multiple kilobases. Extended occupancy patterns appear most prominent at genes encoding crucial developmental regulators and signaling molecules and are highly conserved between Drosophila melanogaster and D. virilis, suggesting functional relevance. Surprisingly, although occupancy is reduced, the Yan monomer still makes extensive multikilobase contacts with chromatin, with an overall pattern similar to that of wild-type Yan. Despite its near-normal chromatin recruitment, the repressive function of the Yan monomer is significantly impaired, as evidenced by elevated target gene expression and failure to rescue a yan null mutation. Together our data argue that SAM-mediated polymerization contributes to the functional output of the active Yan repressive complexes that assemble across extended stretches of chromatin, but does not directly mediate recruitment to DNA or chromatin spreading.

A central goal of microRNA biology is to elucidate the genetic program of miRNA function and regulation. However, relatively few of the effectors that execute miRNA repression have been identified. Because such genes may function in many developmental processes, mutations in them are expected to be pleiotropic and thus are discarded in most standard genetic screens. Here, we describe a systematic screen designed to identify all Drosophila genes in ∼40% of the genome that function in the miRNA pathway. To identify potentially pleiotropic genes, the screen analyzed clones of homozygous mutant cells in heterozygous animals. We identified 45 mutations representing 24 genes, and we molecularly characterized 9 genes. These include 4 previously known genes that encode core components of the miRNA pathway, including Drosha, Pasha, Dicer-1, and Ago1. The rest are new genes that function through chromatin remodeling, signaling, and mRNA decapping. The results suggest genetic screens that use clonal analysis can elucidate the miRNA program and that ∼100 genes are required to execute the miRNA program.