Using Drosophila to uncover molecular and physiological functions of circRNAs.

Circular RNAs (circRNAs) are a class of covalently closed RNA molecules generated by backsplicing. circRNAs are expressed in a tissue-specific manner, accumulate with age in neural tissues, and are highly stable. In many cases, circRNAs are generated at the expense of a linear transcript as back-splicing competes with linear splicing. Some circRNAs regulate gene expression in cis, and some circRNAs can be translated into protein. The advent of deep sequencing and new bioinformatic tools has allowed detection of thousands of circRNAs in eukaryotes. Studying the functions of circRNAs is done using a combination of molecular and genetic methods. The unique genetic tools that can be used in studies of Drosophila melanogaster are ideal for deciphering the functions of circRNAs in vivo. These tools include the GAL4-UAS system, which can be used to manipulate the levels of circRNAs with exquisite temporal and spatial control, and genetic interaction screening, which could be used to identify pathways regulated by circRNAs. Research performed in Drosophila has revealed circRNAs production mechanisms, details of their translation, and their physiological functions. Due to their short lifecycle and the existence of excellent neurodegeneration models, Drosophila can also be used to study the role of circRNAs in aging and age-related disorders. Here, we review molecular and genetic tools and methods for detecting, manipulating, and studying circRNAs in Drosophila.

circMbl functions in cis and in trans to regulate gene expression and physiology in a tissue-specific fashion.

Muscleblind (mbl) is an essential muscle and neuronal splicing regulator. Mbl hosts multiple circular RNAs (circRNAs), including circMbl, which is conserved from flies to humans. Here, we show that mbl-derived circRNAs are key regulators of MBL by cis- and trans-acting mechanisms. By generating fly lines to specifically modulate the levels of all mbl RNA isoforms, including circMbl, we demonstrate that the two major mbl protein isoforms, MBL-O/P and MBL-C, buffer their own levels by producing different types of circRNA isoforms in the eye and fly brain, respectively. Moreover, we show that circMbl has unique functions in trans, as knockdown of circMbl results in specific morphological and physiological phenotypes. In addition, depletion of MBL-C or circMbl results in opposite behavioral phenotypes, showing that they also regulate each other in trans. Together, our results illuminate key aspects of mbl regulation and uncover cis and trans functions of circMbl in vivo.

An in vivo strategy for knockdown of circular RNAs.

Exonic circular RNAs (circRNAs) are highly abundant RNAs generated mostly from exons of protein-coding genes. Assaying the functions of circRNAs is not straightforward as common approaches for circRNA depletion tend to also alter the levels of mRNAs generated from the hosting gene. Here we describe a methodology for specific knockdown of circRNAs in vivo with tissue and cell resolution. We also describe an experimental and computational platform for determining the potential off-target effects as well as for verifying the obtained phenotypes. Briefly, we utilize shRNAs targeted to the circRNA-specific back-splice junction to specifically downregulate the circRNA. We utilized this methodology to downregulate five circRNAs that are highly expressed in Drosophila. There were no effects on the levels of their linear counterparts or any RNA with complementarity to the expressed shRNA. Interestingly, downregulation of circCtrip resulted in developmental lethality that was recapitulated with a second shRNA. Moreover, downregulation of individual circRNAs caused specific changes in the fly head transcriptome, suggesting roles for these circRNAs in the fly nervous system. Together, our results provide a methodological approach that enables the comprehensive study of circRNAs at the organismal and cellular levels and generated for the first time flies in which specific circRNAs are downregulated.

A genome-wide resource for the analysis of protein localisation in Drosophila.

The Drosophila genome contains >13000 protein-coding genes, the majority of which remain poorly investigated. Important reasons include the lack of antibodies or reporter constructs to visualise these proteins. Here, we present a genome-wide fosmid library of 10000 GFP-tagged clones, comprising tagged genes and most of their regulatory information. For 880 tagged proteins, we created transgenic lines, and for a total of 207 lines, we assessed protein expression and localisation in ovaries, embryos, pupae or adults by stainings and live imaging approaches. Importantly, we visualised many proteins at endogenous expression levels and found a large fraction of them localising to subcellular compartments. By applying genetic complementation tests, we estimate that about two-thirds of the tagged proteins are functional. Moreover, these tagged proteins enable interaction proteomics from developing pupae and adult flies. Taken together, this resource will boost systematic analysis of protein expression and localisation in various cellular and developmental contexts.