中研院發現環形RNA 控制多功能幹細胞 耗費4年證實 幹細胞中環形RNA非錯誤 降低遺傳變異腫瘤形成 助癌症藥物發展 2018-1-3 醫藥新聞 可以幫助器官再生的多功能幹細胞，在過去的研究中，
作者：余俊穎博士、李東城博士、吳逸盈博士、葉蟬嫻博士、蔣瑋、
The circular RNA circBIRC6 participates in the molecular circuitry controlling human pluripotency Nature Communications 8, Article number: 1149 (2017) 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.
Introduction Circular RNAs (circRNAs) are closed RNA transcripts, generated by back-splicing of a single pre-mRNA. In this process, the 5 terminus is covalently linked to the 3′terminus, resulting in a scrambled exon order. The first circRNA was identified in the early 1990s1, however, in the subsequent two decades, only a few additional circRNAs were reported1,2,3. These circular transcripts were originally considered errors or byproducts of splicing, but with the emergence of next-generation sequencing (NGS), circRNAs are now known to be abundant and conserved among various biological systems. Moreover, the expression of circRNAs has recently been shown to be tissue specific and regulated in different biological processes, such as epithelial–mesenchymal transition (EMT) and brain development4,5,6. The biogenesis of individual circRNAs must be tightly controlled to produce these expression profiles, and studies on circRNA generation indicate that both cis-elements and trans-acting factors are involved in regulating biogenesis. Cis-elements, such as canonical splicing sites, GU-AG, are necessary for circRNA biogenesis7, and complementary base-pair sequences in the flanking introns (e.g., Alu elements) also contribute to promoting circRNA formation by bringing two distal introns together8, 9. Trans-factors, such as SFs (e.g., Muscleblind and Quaking), interact with the flanking introns of circRNAs to regulate circRNA synthesis5, 10. Thus, it has become clear that the biogenesis of circRNAs is tightly regulated in different biological processes to allow them to fulfill their regulatory roles. However, these regulatory roles are still largely unknown. The biological functioning of circRNAs was first described by Memczak et al.6 and Hansen et al.11, who demonstrated that circRNAs can act as microRNA "sponges" to regulate gene expression. Since then, various functional roles have been reported for circRNAs in controlling biological processes12,13,14, suggesting that circRNAs may have important roles in widespread cellular functions. Human embryonic stem cells (hESCs), which are derived from the pluripotent inner cell mass of preimplantation blastocysts, have the capacity for unlimited self-renewal and pluripotency, giving rise to many cell types in the human body15. A functional analysis of the core pluripotency-associated transcription factors (PATFs), NANOG, OCT4, and SOX2, has revealed that they are indispensable for the maintenance of pluripotency in hESCs16. In addition to transcription factors, non-coding RNAs (ncRNAs), which have little or no protein-coding potential, have also been shown to have important roles in pluripotency maintenance. For example, small ncRNAs (less than 200 nucleotides), such as the microRNAs (miRNAs), miR-302/367, and miR-372 clusters, repress the expression of differentiation-related genes in hESCs17. Conversely, miR-34a and miR-145 are known to repress pluripotency-associated genes to promote in vitro differentiation of hESCs18, 19. In addition, long ncRNAs (lncRNAs; more than 200 nucleotides), such as lncRNA-ES1 and lncRNA-ROR, repress hESC differentiation by recruiting the polycomb repressive complex, PRC2, and inhibiting miRNA activity, respectively20, 21. Furthermore, the trans-spliced lncRNA, tsRMST, has recently been demonstrated to promote the undifferentiated status of hESCs by repressing early lineage-associated transcription factors22 and WNT signaling23 through the PRC2 complex. These studies clearly establish the importance of ncRNAs in pluripotency maintenance; however, a functional role for circRNAs in pluripotency status has not been previously reported. To explore the functional roles of circRNAs in regulating human pluripotency, we identified and validated a subset of hESC-enriched circRNAs. Through gain-of-function and loss-of-function experiments, we further demonstrated that two circRNAs, circBIRC6 and circCORO1C, are functionally associated with pluripotency maintenance and reprogramming. Furthermore, we showed that circBIRC6 is enriched in the RNA-induced silencing complex (RISC), containing the catalytic subunit AGO2, and promotes the pluripotent state by inhibiting miR-34a-mediated and miR-145-mediated suppression of NANOG, OCT4, and SOX2 expression. Studies on circBIRC6 biogenesis showed that the pluripotency-associated genes NANOG and OCT4 regulate expression of the SF ESRP1 (epithelial-splicing regulatory protein 1), which is responsible for the generation of circBIRC6 in hESCs. Collectively, our results demonstrate that circRNAs participate in the molecular circuitry that controls human pluripotency.
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