Ananya Mahapatra, Alfa Dhakal, Aika Noguchi, Pranathi Vadlamani, Heather A. Hundley
The ability to alter gene expression programs in response to changes in environmental conditions is central to the ability of an organism to thrive. For most organisms, the nervous system serves as the master regulator in communicating information about the animal’s surroundings to other tissues. The information relay centers on signaling pathways that cue transcription factors in a given cell type to execute a specific gene expression program, but also provide a means to signal between tissues. The transcription factor PQM-1 is an important mediator of the insulin signaling pathway contributing to longevity and the stress response as well as impacting survival from hypoxia. Herein, we reveal a novel mechanism for regulating PQM-1 expression specifically in neural cells of larval animals. Our studies reveal that the RNA-binding protein (RBP), ADR-1, binds to pqm-1 mRNA in neural cells. This binding is regulated by the presence of a second RBP, ADR-2, which when absent leads to reduced expression of both pqm-1 and downstream PQM-1 activated genes.
Aerobic heterotrophs need to obtain nutrition and oxygen from the environment, the prolonged absence of which can lead to undesirable consequences including death. However, fluctuations in oxygen and nutrient availability are common in nature and during development; thus, organisms must have a means to both sense the environment and respond. At the most extreme, animals can effectively halt developmental and cellular programs resulting in a transient quiescent state . For example, in the model organism Caenorhabditis elegans (C. elegans), the absence of oxygen can lead to a state of “suspended animation” , while first larval stage (L1) animals hatched in the absence of food enter a state of halted development commonly referred to as “L1 arrest” [3,4].
Materials and methods
C. elegans strains and maintenance
All worms were maintained under standard laboratory conditions on nematode growth media seeded with Escherichia coli OP50 . The following previously generated strains were used in this study: Bristol strain N2, BB19 (adr-1(tm668)) , BB20 (adr-2(ok735)) , BB21 (adr-1(tm668);adr-2(ok735)) , HAH22 (adr-2(gk777511)  agIs6[dod-24::GFP] , daf-2(m596) , pqm-1(ok485) . Neural cells were isolated from HAH45 (prab3::rfp::C35E7.6 3′ UTR; prab3::gfp::unc-54 3′ UTR; unc-119 genomic rescue), HAH46 (adr-2(ok735); prab3::rfp::C35E7.6 3′ UTR; prab3::gfp::unc-54 3′ UTR; unc-119 genomic rescue) and BB79 (adr-1(tm668);adr-2(ok735); prab3::rfp::C35E7.6 3′ UTR; prab3::gfp::unc-54 3′ UTR; unc-119 genomic rescue) .
Decreased expression of genes regulated by insulin signaling upon loss of adr-2
As a first step towards addressing whether ADR-2 regulates insulin signaling, the transcriptomes of wild type and adr-2-deficient animals were compared. As editing of daf-2 was observed in neural cells isolated from synchronized L1 animals , differential gene expression was analyzed in RNA isolated from these same types of biological samples. Using datasets from previously performed RNA-sequencing (RNA-seq) of 3 biological replicates of wild type and adr-2(-) neural cells from synchronized L1 animals , differential gene expression analysis identified 697 genes significantly altered in neural cells from adr-2(-) animals (p value < 0.05 and log2fold change > |0.5|), with nearly 3 times as many down-regulated genes (501) as up-regulated genes (196) (Fig 1A, S1 Table). These misregulated genes were subjected to gene set enrichment analysis using a C. elegans specific software, WormCat .
In these studies, we determined the tissue-specific contributions of ADAR proteins in regulating the insulin signaling pathway in C. elegans. Our data revealed unique ADR-1 RNA binding that occurs in the nervous system specifically in the absence of adr-2. Furthermore, our neural cell data indicate that the binding of ADR-1 in neural cells is sufficient to cause down-regulation of pqm-1 transcript in the absence of adr-2. However, the molecular details of how ADR-1 binding leads to decreased pqm-1 expression are an open question. Previous work from our lab has shown that ADR-1 binding to another transcript, clec-41, is important to promote neural gene expression ; however, that mechanism was editing and ADR-2 dependent. Editing-independent effects of ADARs on mRNA stability have been identified for human ADAR1 and ADAR2 [50,51].
We thank Christiane Hassel (IUB- Flow Cytometry Core Facility) for assisting in COPAS sorting of transgenic animals and isolation of neural cells. We thank Dr. Andras Kun (IUB- Light Microscopy Imaging Center) for the training and facilitating usage of the confocal microscope. We thank current members of the Hundley lab, Dr. Chinnu Salim, Boyoon Yang, Emily Erdmann, Mary Skelly, and former Hundley lab member Dr. Reshma Raghava Kurup for careful reading of the manuscript. We thank graduate student Shefali Shefali for her tremendous help in taking the confocal images and Boyoon Yang for assisting in the masking of genotypes for the survival assays.
Citation: Mahapatra A, Dhakal A, Noguchi A, Vadlamani P, Hundley HA (2023) ADAR-mediated regulation of PQM-1 expression in neurons impacts gene expression throughout C. elegans and regulates survival from hypoxia. PLoS Biol 21(9): e3002150. https://doi.org/10.1371/journal.pbio.3002150
Academic Editor: Wendy V. Gilbert, Yale University, UNITED STATES
Received: April 27, 2023; Accepted: August 23, 2023; Published: September 25, 2023
Copyright: © 2023 Mahapatra et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by the National Science Foundation (Award 191750 to HAH), National Institute of Health/National Institute of General Medical Sciences (R01 GM130759 to HAH) and the John R. and Wendy L. Kindig Fellowship (to AM). Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: cDNA, complementary DNA; ChIP, chromatin immunoprecipitation; ILP, insulin-like peptide; RBP, RNA-binding protein; RIP, RNA immunoprecipitation; RNAi, RNA interference; UTR, untranslated region