Circular IRE-type RNAs of the NR5A1 gene are formed in adrenocortical cells

Highlights

• First report of tissue-specific transcriptional response to IRE1α via circular RNAs connected by IRE1α cleavage sites

Abstract

The recently discovered circular RNAs (circRNAs) are mostly formed by back-splicing where the downstream 5′ splice site splices to the upstream 3’ splice site by conventional pre-mRNA splicing. These circRNAs regulate gene expression by acting as sponges for micro-RNAs or RNA-binding proteins. Here we show that the NR5A1 (previously called Ad4BP or SF-1) gene which is exclusively expressed in the adrenal cortex and steroidogenic tissue can form atypical circRNAs by unconventional splicing. Two stem loops with inositol-requiring protein-1α (IRE1α) cleavage sites are connected by an IRE1α cleavage site to form a circRNA (circIRE RNA). From total RNA of normal human adrenal cortex, we detected a circIRE RNA with connected ends by IRE1α cleavage sites in exon 6 and exon 1 (circIRE NR5A1 ex6-1 RNA). circIRE NR5A1 ex6-1 RNA was not detected in the adrenocortical cancer cell line, H295R. When IRE1α was expressed in H295R cells a different circIRE NR5A1 RNA connecting IRE1-cleavage sites in exon 7 and exon 1 was detected (circIRE NR5A1 ex7-1 RNA). The expression of this circIRE RNA was inhibited by the IRE1 inhibitor 1, STF-083010, implicating that it was formed via the ER stress pathway, where IRE1α is a major factor. This is the first report of this type of circular RNA connected by IRE1-cleavage sites found to be expressed in mammalian cells in a tissue-specific manner. To our surprise, the concomitant expression of NR5A1 was increased by IRE1α implicating that NR5A1 was not subjected to IRE1-dependent decay of mRNA (RIDD) but rather activating a transcriptional regulatory network to cope with ER stress in steroidogenic tissue reminiscent to XBP1 in other tissue. We believe this is the first report of such tissue-specific transcriptional cascade responding to ER stress as well as the novel finding of circular RNAs connected by IRE1α cleavage sites expressed in mammalian tissue.

Introduction

Two recent analyses of circular RNAs (circRNAs) by previously published RNA-seq data reported that 11.9% [1] and 36.97–50.04% [2] of circRNAs are tissue-specific in human adult tissue. The expression of circRNAs are more abundant in fetal than in adult tissue and in gland tissue than in other tissue with 2311 circRNAs found in the adrenal gland [2]. From functional enrichment analysis, these tissue-specific circRNAs bind RNA-binding proteins and micro-RNAs to regulate tissue development and differentiation [1]. Most circRNAs are formed by back-splicing using conventional splicing from the downstream 5′ splice site to the upstream 3’ splice site [3]. Besides conventional splicing, tRNAs are known to be spliced [4] by recognizing RNA secondary structure [5]. tRNA splicing is not completely conserved between archea and eukarya [6] and circular RNA formation of this type (tRNA intronic circular RNAs (tricRNAs)) have not yet been detected endogenously but can be engineered in cultured cells [6].

An unconventional type of splicing, very similar to tRNA splicing, is found in the unfolded protein response (UPR) pathway following endoplasmic reticulum (ER) stress. ER stress is known to activate three different molecules, inositol-requiring protein-1 (IRE1), activating transcription factor-6 (ATF6), and protein kinase RNA (PKR)-like ER kinase (PERK), to transmit the information to the cytoplasm. The transmembrane kinase Ire1p was found to cleave HAC1 mRNA and ligate the two ends by tRNA ligase, a key step of the unfolded protein response in yeast Saccharomyces cerevisiae [7]. Likewise, this mechanism was also found in the UPR of metazoans where IRE1 cleaves XBP-1 mRNA [8]. IRE1 is unique in that it functions as a protein kinase and a site-specific endoribonuclease [9]. IRE1 recognizes and cuts an RNA target sequence (consensus: 5′-CUGCAG-3′) [10] on mRNA at two sites and ligate the two fragments by a specific tRNA ligase in mammals removing the sequence in between [11]. The non-XBP1 targets of IRE1 have characteristics to preserve homeostasis or induce cell death, that is different from XBP1 where a transcriptional network is activated to cope with ER stress. The non-XBP-1 targets are mainly degraded by IRE1 through the IRE1-dependent decay of mRNA (RIDD) pathway [12,13]. More than thirty genes are known to be subjected to RIDD [[12], [13], [14], [15], [16], [17]].

We sought to check circRNAs of the human NR5A1 gene [18], the major regulator of development, differentiation as well as steroidogenesis of the adrenal gland [[19], [20], [21]]. We focused on whether the excised fragment of RIDD may form circRNAs. Since circRNA formation have the tendency to form with long exons [22], we initiated our study by analyzing circRNAs containing NR5A1 exon 4.