The circular RNA circBIRC6 participates in the molecular circuitry controlling human pluripotency

Chun-Ying Yu,Ching-Yu Chuang,Wei Chiang,Tung-Cheng Li,Yi-Ying Wu,Chan-Hsien Yeh,Hung-Chih Kuo

doi:10.1038/s41467-017-01216-w

Abstract

Accumulating evidence indicates that circular RNAs (circRNAs) are abundant in the human transcriptome. However, their involvement in biological processes, including pluripotency, remains mostly undescribed. We identified a subset of circRNAs that are enriched in undifferentiated human embryonic stem cells (hESCs) and demonstrated that two, circBIRC6 and circCORO1C, are functionally associated with the pluripotent state. Mechanistically, we found that circBIRC6 is enriched in the AGO2 complex and directly interacts with microRNAs, miR-34a, and miR-145, which are known to modulate target genes that maintain pluripotency. Correspondingly, circBIRC6 attenuates the downregulation of these target genes and suppresses hESC differentiation. We further identified hESC-enriched splicing factors (SFs) and demonstrated that circBIRC6 biogenesis in hESCs is promoted by the SF ESRP1, whose expression is controlled by the core pluripotency-associated factors, OCT4 and NANOG. Collectively, our data suggest that circRNA serves as a microRNA “sponge” to regulate the molecular circuitry, which modulates human pluripotency and differentiation.

Highlights

Accumulating evidence indicates that circular RNAs are abundant in the human transcriptome
To identify a pool of potential circRNAs that may be expressed in human embryonic stem cells (hESCs), we utilized a human circRNA database that comprises candidates identified from various cell types[6], and selected 61 strong candidates for circRNAs that had more than 40 reads supporting a circular junction
We further confirmed the presence of transcripts for circBIRC6, circCORO1C, and circMAN1A2, which exhibited the highest association with pluripotency status, in hESCs by Northern blot analysis using probes that target the circular junction of these circRNAs (Supplementary Fig. 1)

Summary

Results

ICC analyses revealed a decreased number of NANOG+ and OCT4+ cells, and an increased number of Brachyury+ and SOX17+ cells in shcircB-2transduced hESCs (Supplementary Fig. 3f), suggesting that circBIRC6, but not linear BIRC6, is critical for pluripotency maintenance. These results indicate that circBIRC6 and circCORO1C may be functionally involved in the maintenance of hESC pluripotency. ICC analysis revealed an increased number of NANOG+ and OCT4+ cells in differentiated H9-circB and H9-circC, and a decreased number of Brachyury+, SOX17+, and PAX6+ cells (Fig. 3e, f) These results show that circBIRC6 and circCORO1C, but not circMAN1A2, maintain pluripotency marker expression in differentiated hESCs. Expressing circBIRC6 or circCORO1C promotes reprogramming. RNA immunoprecipitation (RIP) assays further showed that circBIRC6, but not circCORO1C or either linear isoform, was

H9-circB EB

Discussion

ESRP1-Luc 4 ESRP1-m3-Luc

Methods