Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor

1370–1383 Nucleic Acids Research, 2016, Vol. 44, No. 3 doi: 10.1093/nar/gkv1367 Published online 10 December 2015 Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor Yehoshua Enuka, Mattia Lauriola, Morris E. Feldman, Aldema Sas-Chen, Igor Ulitsky and Yosef Yarden* Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel Received July 24, 2015; Revised November 22, 2015; Accepted November 24, 2015 ABSTRACT INTRODUCTION Growth factors are evolutionarily conserved molecules, which are secreted by specific cells and bind specific receptors on the surface of target cells (1). One family of growth factors comprises 11 epidermal growth factor- (EGF-) like molecules. These ligands regulate proliferation and migration of epithelial and other cell lineages throughout embryonic development and in adulthood, such as in mammary gland development (2). Importantly, growth factors often induce rapid effects on signaling pathways, but their longterm biological effects, such as cell cycle regulation and chemotaxis, require synthesis of new RNAs and proteins (3). Correspondingly, EGF family growth factors induce wave-like bursts of transcription of distinct RNA molecules, * To whom correspondence should be addressed. Tel: +972 8 934 3974; Fax: +972 8 934 2488; Email:  C The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Circular RNAs (circRNAs) are widespread circles of non-coding RNAs with largely unknown function. Because stimulation of mammary cells with the epidermal growth factor (EGF) leads to dynamic changes in the abundance of coding and non-coding RNA molecules, and culminates in the acquisition of a robust migratory phenotype, this cellular model might disclose functions of circRNAs. Here we show that circRNAs of EGF-stimulated mammary cells are stably expressed, while mRNAs and microRNAs change within minutes. In general, the circRNAs we detected are relatively long-lived and weakly expressed. Interestingly, they are almost ubiquitously co-expressed with the corresponding linear transcripts, and the respective, shared promoter regions are more active compared to genes producing linear isoforms with no detectable circRNAs. These findings imply that altered abundance of circRNAs, unlike changes in the levels of other RNAs, might not play critical roles in signaling cascades and downstream transcriptional networks that rapidly commit cells to specific outcomes. starting with a group of immediate early genes (IEGs) and culminating in a large group of fate-determining mRNAs (4). Like mRNAs, specific microRNAs display dynamic upand down-regulation in response to growth factors. For example, a group of immediately down-regulated microRNAs (ID-miRs) normally suppresses transcription of the group of IEGs (5). Another type of non-coding RNAs, long non-coding RNAs (lncRNAs), is similarly regulated by growth factors. For example, lncRNA-ATB is activated by the ␤ type transforming growth factors (TGF-␤), to promote invasion of hepatocytes (6). Although synthetic circular RNAs have the ability to produce a protein product in vitro (7), in general, natural circRNAs are believed to be non-coding (8,9). Whether or not circRNAs are dynamically regulated following stimulation with growth factors is currently unknown. Circularization of RNAs was recently recognized to broadly expand the transcriptome (8– 18). CircRNAs in animals have been discovered more than 30 years ago, but they were largely neglected due to rarity and lack of function. Due to the advent of next generation sequencing, thousands of different circRNAs were recently identified in various organisms, from archaea to human (9,14–16,19,20). The circRNA CDR1as was found to contain an exceptionally high number of binding sites specific to a miRNA and indeed was found to antagonize miRNA activity by a sponge-like mechanism (15,21), which led to the notion that circRNAs may function to sequester miRNAs (22–26). A recent study, however, raised doubts regarding a biological function of most circRNAs (27). Sponge-like recruitment of multiple microRNA would be an attractive mechanism in the context of growth factor activation. Interestingly, analysis of epithelial cells that underwent an epithelial-mesenchymal transition (EMT), after a 21 day-long treatment with TGF-␤, revealed that hundreds of circRNAs were regulated during this process (28). Additional reports imply that circRNAs are functional molecules, rather than by-products of mis-splicing. For example, treatment of endothelial cells with tumor necrosis factors revealed that circRNA formation correlates with exon skipping (29), and yet another report has shown that Nucleic Acids Research, 2016, Vol. 44, No. 3 1371 MATERIALS AND METHODS Cell culture and metabolic labeling MCF10A cells were cultured as described (32) in DME:F12 medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10 ␮g ml−1 insulin, 0.1 ␮g ml−1 cholera toxin, 0.5 ␮g ml−1 hydrocortisone, 5% heat-inactivated horse serum (Biological Industries, Beit-Haemek, Israel) and 10 ng ml−1 EGF. For time course experiments, cells were starved overnight in medium without additives, and thereafter stimulated with EGF (10 ng ml−1 ) or dexamethasone (DEX; 100 nM). RNA metabolic labeling was performed in different concentrations of 4-thiouridine (4sU; Sigma), as recommended before (35). The labeling reagent was added to the medium (0.5 mM final concentration) and incubated with cells for 20 min. This was followed by 40 min of labeling (0.3 mM) and further incubations for 1, 2 or 4 h (0.2 mM). When treated with EGF, cells were concomitantly labeled with 4sU. Sequencing of RNA derived from MCF10A cells Total RNA was isolated from MCF10A cells using Trizol (Invitrogen). Ribosomal RNA was removed using the Ribominus kit (Invitrogen). A cDNA library was generated and sequenced using an Illumina Hiseq 2500 (1 × 100 bp runs), as instructed by Illumina’s RNA-seq protocols. Antisense (15). Our circRNA analyses conditioned that at least two independent reads support existence of a noncanonical sequence junction. mRNA and miRNA expression data from microarrays Expression data of mRNA and miRNA were previously generated in our lab using MCF10A cells and a stimulation protocol identical to the one used in the current study (5,36). We randomly selected 16.1% of mRNAs to obtain the same coverage as we obtained by high-throughput polymerase chain reaction (PCR) for circRNAs identified by sequencing (241 out of 1498; 16.1%). Thereafter, the selected mRNAs were sorted according to their maxim (...truncated)