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A circular RNA Edis-Relish-castor axis regulates neuronal development in Drosophila [1]

['Wei Liu', 'Department Of Medicine', 'Johns Hopkins University School Of Medicine', 'Baltimore', 'Maryland', 'United States Of America', 'Department Of Biological Chemistry', 'Department Of Oncology', 'Cancer', 'Blood Disorders Institute']

Date: 2022-12 

Circular RNAs (circRNAs) are a new group of noncoding/regulatory RNAs that are particularly abundant in the nervous system, however, their physiological functions are underexplored. Here we report that the brain-enriched circular RNA Edis (Ect4-derived immune suppressor) plays an essential role in neuronal development in Drosophila. We show that depletion of Edis in vivo causes defects in axonal projection patterns of mushroom body (MB) neurons in the brain, as well as impaired locomotor activity and shortened lifespan of adult flies. In addition, we find that the castor gene, which encodes a transcription factor involved in neurodevelopment, is upregulated in Edis knockdown neurons. Notably, castor overexpression phenocopies Edis knockdown, and reducing castor levels suppresses the neurodevelopmental phenotypes in Edis-depleted neurons. Furthermore, chromatin immunoprecipitation analysis reveals that the transcription factor Relish, which plays a key role in regulating innate immunity signaling, occupies a pair of sites at the castor promoter, and that both sites are required for optimal castor gene activation by either immune challenge or Edis depletion. Lastly, Relish mutation and/or depletion can rescue both the castor gene hyperactivation phenotype and neuronal defects in Edis knockdown animals. We conclude that the circular RNA Edis acts through Relish and castor to regulate neuronal development.

Circular RNAs (circRNAs) are a new group of noncoding/regulatory RNAs that are particularly abundant in the nervous system, although their physiological functions are underexplored. Here we report that the brain-enriched circular RNA Edis (Ect4-derived immune suppressor) plays an essential role in neuronal development in the fruitfly Drosophila melanogaster, as its depletion causes defects in the development of neurons in a brain structure called mushroom body (MB), as well as impaired locomotor activity and shortened lifespan of adult flies. In addition, we show that the castor gene, which encodes a protein involved in neurodevelopment, is upregulated in Edis knockdown neurons. Notably, flies with increased levels of castor or reduced levels of Edis display similar phenotypes, and reducing castor levels rescues the developmental defects in Edis-depleted neurons. Furthermore, we find that the immune regulator Relish occupies a pair of sites at the castor gene locus, and that both sites are required for optimal castor gene activation by either immune challenge or Edis depletion. Lastly, Relish mutation and/or depletion can rescue both the castor gene hyperactivation phenotype and neuronal defects in Edis knockdown animals. We conclude that the circular RNA Edis acts through Relish and castor to regulate neuronal development.

Funding: This work was supported by the American Heart Association grant 16GRNT31360017 (to R.Z., https://www.heart.org/en/professional/institute/grants ); National Institutes of Health grants 1R01AI140049 and 1R21AI131099 (to R.Z., www.nih.gov ); and start-up funds from the Sanford Burnham Prebys Medical Discovery Institute ( https://www.sbpdiscovery.org/ ) and Johns Hopkins University (to R.Z., https://www.hopkinsmedicine.org/som/ ). J.L. is supported by the Intramural Research Program of the NIEHS/NIH ( https://www.niehs.nih.gov/research/atniehs/dir/index.cfm ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we report that Edis is critically required for mushroom body (MB) development in Drosophila. We show that the Edis transcript is enriched in neurons, consistent with its role in neuronal development. Notably, loss of Edis leads to axonal misguidance in MB neurons and elevated expression of castor, which encodes a transcription factor critical for neuronal development. Importantly, overexpression of castor phenocopies Edis depletion, and reducing castor levels in Edis-depleted neurons rescues defects in MB morphology, locomotor activity and lifespan. We provide evidence that the immune transcription factor Relish binds to the castor promoter and regulates castor transcription. Thus, our study reveals a crucial function of the circRNA Edis in regulating MB neuronal development in Drosophila, establishes castor as an effector/target gene downstream of Edis, and generates an animal model that can facilitate unraveling of intricate interplay between innate immunity signaling and neuronal development.

In a recent study, we describe the identification and functional characterization a circRNA circEct4, also known as Edis (Ect4-derived immune suppressor) [ 39 ]. Knockdown of Edis, but not its linear sibling Ect4, specifically in neurons causes hyperactivation of innate immunity and myriad defects in neuronal development. We show that Edis can be translated into a functional protein Edis-p, which binds to, and compromises, the proteolytic processing/activation of the immune transcription factor Relish. In addition, inactivation of Relish in Edis-depleted neurons rescues the innate immunity hyperactivation phenotype, suggesting that Relish acts downstream of Edis to regulate immunity. However, the detailed mechanism underlying the function of Edis in regulating the neurodevelopment is still elusive, and the target/effector gene(s) downstream Edis/Relish remain to be identified and functionally characterized.

Drosophila melanogaster is a powerful model organism to advance our understanding of the molecular mechanism underlying innate immunity activation. Upon encountering diaminopimelic acid (DAP)-type peptidoglycan (PGN), a cell wall component derived from Gram-negative and certain Gram-positive bacteria, a dedicated IMD (immune deficiency) signaling pathway is activated [ 32 ]. The IMD pathway involves membrane-bound receptor PGRP-LC; adaptor molecules IMD and dFADD; ubiquitination enzymes Bendless, dUEV1a and dIAP-2; protein kinase complexes dTAK1/dTAB2 and Ird5/Kenny; and the caspase Dredd, culminating in the proteolytic processing and activation of the NF-κB family transcription factor Relish, nuclear translocation of the N-terminal fragment of Relish, and activation of genes encoding a battery of antibacterial peptides [ 33 – 36 ]. Similar to the observations made in humans, aberrant activation of innate immunity in Drosophila can result in phenotypes indicative of neurodegeneration. For example, depletion of ATM (AT mutated) in glial cells causes elevated expression of innate immune response genes in glial cells as well as neuronal and glial cell death, and a reduction in mobility and longevity [ 37 ]. In addition, it has been reported that flies with mutations in dnr1 (defense repressor 1) exhibit shortened lifespan and progressive, age-dependent neuropathology associated with aberrant activation of the IMD pathway and elevated expression of antimicrobial peptide (AMP) genes [ 38 ]. Furthermore, ectopic expression of individual AMP genes in the Drosophila brain results in brain damage [ 38 ]. These findings highlight the connection between dysregulated innate immunity signaling and neurodegeneration in flies.

Innate immunity, the first line of defense, protects hosts against invading microbes and adverse effect of stress signals generated by injured cells. While rapid and robust activation of the innate immune response is crucial for host fitness, aberrant or prolonged activation can cause detrimental consequences. For example, aging and neurodegenerative conditions are often associated with aberrant activation of immunity signaling [ 29 ]. In addition, long-term pharmacological suppression of the inflammatory response can lead to a reduction in risk of developing neurodegenerative diseases [ 30 , 31 ]. Thus, the magnitude and duration of innate immunity activation need to be tightly controlled in order to maintain a delicate balance between host defense and nervous system integrity/function.

While circRNAs are present across most cell/tissue types [ 19 – 21 ], they are particularly abundant in the nervous system [ 3 , 22 – 26 ], suggesting a key role in neurodevelopment. Indeed, a handful of neuronal circRNAs have been functionally characterized. For example, CDR1as binds to microRNA-7 (miR-7) and regulates miR-7 biogenesis, thereby impacting brain development [ 7 , 8 , 12 , 27 ]. In addition, circZNF827 functions as a scaffold for a transcription repressive complex containing ZNF827, hnRNP K, and hnRNP L to regulate neuronal differentiation [ 28 ]. Furthermore, the psychiatric disease-associated circRNA circHomer1a interacts with the HuD protein and further influences HuD gene expression in the frontal cortex [ 13 ]. Despite these well-characterized circRNAs, our knowledge of how circRNAs are involved in neuron development/function is still limited. Thus, it is important to identify and functionally characterize additional neuronal circRNAs in healthy and disease settings, and to elucidate the underlying mechanisms.

Circular RNAs (circRNAs) are the latest addition to the noncoding and regulatory RNA collection. They are characterized as covalently closed RNA loops generated by “head-to-tail” back-splicing events [ 1 , 2 ]. With the development of high-throughput sequencing technologies and bioinformatic approaches, thousands of circRNAs have been identified in a wide variety of eukaryotic organisms including human, mouse, worm, and fruit fly [ 3 – 6 ]. Subsequent functional studies have implicated select circRNAs in various physiological and pathological processes, including testes development [ 7 , 8 ], cell cycle progression [ 9 ], cancer-associated cell proliferation [ 10 , 11 ], and neuropsychiatric disorders [ 12 , 13 ]. Circular RNAs can function as regulators of microRNA biogenesis/function, operate as scaffold for the assembly of protein/RNA complexes, or modulate host gene expression. Recently, select circRNAs have been shown to encode functional proteins [ 14 , 15 ]. Since circRNAs generally lack a 5’ cap and poly(A) tail, translation of circRNAs is mediated by cap-independent mechanisms, including internal ribosome entry site (IRES) or N6-methyladenosine (m6A) mediated ribosome recruitment [ 1 , 16 , 17 , 18 ].

Results

The neurodevelopmental phenotypes elicited by Edis depletion depend on Relish We found that Edis depletion in the Drosophila CNS leads to activation of the IMD innate immunity signaling pathway, with dramatically elevated expression of several AMP genes that are normally regulated by Relish, a key immune transcription factor (S2 Fig). To investigate the relationship between the neuronal defects and the immunity hyperactivation phenotypes, we examined the impact of Relish mutation on the neuronal phenotypes of Edis knockdown animals. Consistent with recent reports that implicate Relish in neurodevelopment [54,55], we found that about a third of Relish null mutants display β lobe fusion phenotypes (Fig 3A and 3B). Despite of this, in Relish null mutant background, the MB morphology phenotype in Elav>shEdis flies was (at least partially) rescued (Fig 3A and 3B, compare RelE20/E38 with RelE20/+, RelE38/+ or +/+ genetic background). Importantly, a similar observation was made upon depletion of Relish in neurons (Fig 3C and 3D), demonstrating a cell autonomous interaction between Relish and Edis in regulating neurodevelopment. Lastly, the lifespan phenotype was also affected by Relish mutation (Fig 3E). We note that the lifespan differences are complex phenotypes and most likely do not result from Kenyon cell alterations, and that lifespan of Edis knockdown flies was also affected by genetic background (Fig 3E, compare Rel+/+ with RelE20/+ and RelE38/+). Nonetheless, taken together, data from our analysis on MB morphology in various combinations of Edis and Relish mutant/knockdown backgrounds strongly suggest that the neurodevelopmental phenotypes elicited by Edis depletion depend (at least partially) on Relish. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Neuronal phenotypes elicited by Edis depletion depend on Relish. (A) Confocal images of dorsal anterior regions of adult brains with various MB morphology phenotypes. Depletion of Edis in neurons resulted in a spectrum of severe morphological defects in the MBs as noted above each panel. The scale bar indicates 50 μm. (B) MB morphology phenotypes were quantified in flies with the neuron-specific expression of control shgfp or shEdis driven by the Elav-Gal4 driver in wildtype, Relish heterozygous (RelE20/+ or RelE38/+) or homozygous (RelE20/E38) mutant background. Chi-squared test was employed in statistical analysis. Sample numbers and percentages of samples showing normal MB morphology in each genotype are shown. (C-D) MB morphology phenotypes of in flies with neuron-specific expression of various combinations of shRNA transgenes (control shgfp, shEdis and shRel) driven by the Elav-Gal4 driver are shown in C and quantified in D. Chi-squared test was employed in statistical analysis. Sample numbers and percentages of samples showing normal MB morphology in each genotype are shown. (E) Lifespan of flies of select genotypes in B is shown. https://doi.org/10.1371/journal.pgen.1010433.g003

Castor is upregulated upon neuronal Edis depletion It has been reported that microbial infection or ectopic expression of AMP genes can lead to neurodegeneration [38]. To examine whether forced expression of AMP genes in the brain can lead to defects in MB morphology, we expressed individual AMP genes, including Diptericin A (DptA), Drosocin (Dro), Defensin (Def), and Drosomycin (Drs) in neurons using Elav-Gal4. Interestingly, while a fraction of animals with ectopic expression of any of the four AMP genes displayed defective MB morphology, the phenotype is far milder than that seen in Elav>shEdis animals, as only 10–18% Elav>AMP animals showed MB morphology defects (Fig 4A–4F). In addition, only β lobe fusion, but no missing αβ lobe phenotypes were observed in Elav>AMP animals (Fig 4A–4F). Given that both innate immunity hyperactivation and MB morphology phenotypes elicited by neuronal Edis depletion are suppressed in flies carrying mutations in Relish, our data strongly suggest the presence of additional Edis target/effect gene(s) beside AMPs that act downstream of Relish to regulate MB morphology. To search for these gene(s), we performed RNA-seq analysis using Edis-depleted and control brain tissues, from which we identified a total of 777 transcripts that displayed significant changes in RNA levels upon Edis knockdown (412 upregulated and 365 downregulated) (S1 Table and Fig 4G). Notably, several AMP genes (i.e., CecA1, CecA2, CecB, CecC, DptA, AttC, Mtk, Dro and Drs) were among the group of upregulated genes (Fig 4G), thereby validating our approach. The significantly changed genes could be grouped into 16 functional categories based on their predicted/validated roles using Gene Ontology (GO) (Fig 4H). Among these groups of genes, four are related to immune responses (Fig 4H), in addition to genes implicated in nervous system development. As flies missing neuronal Edis display profound neurodevelopmental phenotypes, we selected 17 neurodevelopment-related genes and performed RT-qPCR to examine their expression level in shEdis brain tissues. We note that not all of the 17 genes have scored in our RNA-seq analysis. Among the genes analyzed, castor was the most significantly activated in Edis knockdown brain (Fig 4I). Castor encodes a transcription factor that is expressed in late stages of embryonic neuroblast lineages, and has been shown to be involved in MB development [56], raising an intriguing possibility that dysregulation of castor expression by Edis depletion might be (at least partially) responsible for the MB morphology phenotypes in the CNS. PPT PowerPoint slide

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TIFF original image Download: Fig 4. castor is upregulated upon neuronal Edis depletion. (A-D) Images showing typical MB morphologies in flies with individual AMP overexpression. The scale bar indicates 50 μm. (E) MB morphology phenotypes were quantified in flies with overexpression of AMP genes DptA, Drs, Dro, and Def in neurons. Sample numbers and percentages of samples showing normal MB morphology in each genotype are shown. (F) MB morphology phenotypes were quantified in flies with the indicated genotypes (neuron-specific expression of control shgfp, shEdis or AMP genes driven by the Elav-Gal4 driver). Note that the shgfp and shEdis data are identical to those shown in Fig 1I. We consolidated the individual data points from F as Elav>AMPs. Chi-squared test was employed in statistical analysis. Sample numbers and percentages of samples showing normal MB morphology in each genotype are shown. (G) A volcano plot of gene expression profile of Elav>shEdis brain samples compared to wild type. Significantly up- (red, p ≤ 0.05 and logFC > 1) and down-regulated genes (green, p ≤ 0.05 and logFC ≤ -1) are shown. (H) The significantly changed genes were subjected to gene ontology (GO) enrichment analysis. Shown are major biological processes they are involved in. (I) RNA levels of select genes involved in neurodevelopment were measured by real-time PCR in Elav>shEdis brains and compared with control. castor is significantly upregulated in Edis depleted brain tissue (n = 3). https://doi.org/10.1371/journal.pgen.1010433.g004

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010433

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