XIST sponges miR-320d to promote chordoma progression by regulating ARF6.

Background: Long non-coding RNAs (lncRNAs) have been demonstrated to play important roles in various tumors, including chordoma. The purpose of this study was to investigate the role and mechanism of lncRNA X-inactive specific transcript (XIST) in chordoma. Methods: RNA levels and protein levels were measured by real-time quantitative polymerase chain reaction (RT­qPCR) and western blot assay, respectively. Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay and colony formation assay. Tanswell assay was used to examine cell migration and invasion. Cellular glycolysis was examined via the measurement of extracellular acidification rate (ECAR) and lactate production. The interaction between microRNA-320d (miR-320d) and XIST or ADP-ribosylation factor 6 (ARF6) was predicted by bioinformatics analysis and verified by a dual-luciferase reporter and RNA-pull down assays. The xenograft tumor model was used to explore the biological function of XIST in vivo. Results: XIST was overexpressed in chordoma tissues. XIST knockdown suppressed chordoma cell proliferation, migration, invasion, and glycolysis. XIST acted as a sponge of miR-320d. Moreover, miR-320d overexpression inhibited the proliferation, migration, invasion, and glycolysis of chordoma cells. ARF6 was a direct target of miR-320d, and XIST upregulated ARF6 expression via sponging miR-320d. Furthermore, overexpression of ARF6 reversed the inhibitory effects of XIST knockdown on chordoma cell proliferation, migration, invasion, and glycolysis. Importantly, XIST silencing blocked xenograft tumor growth in vivo. Conclusion: XIST knockdown inhibited chordoma progression via regulating the miR-320d/ARF6 axis, providing a novel insight into chordoma pathogenesis.

CsMYB3 and CsRuby1 form an 'Activator-and-Repressor' Loop for the Regulation of Anthocyanin Biosynthesis in Citrus.

Anthocyanins are preferentially accumulated in certain tissues of particular species of citrus. A R2R3-MYB transcription factor (named Ruby1) has been well documented as an activator of citrus anthocyanin biosynthesis. In this study, we characterized CsMYB3, a transcriptional repressor that regulates anthocyanin biosynthesis in citrus. CsMYB3 was expressed in anthocyanin-pigmented tissues, and the expression was closely associated with that of Ruby1, which is a key anthocyanin activator. Overexpression of CsMYB3 in Arabidopsis resulted in a decrease in anthocyanins under nitrogen stress. Overexpression of CsMYB3 in the background of CsRuby1-overexpressing strawberry and Arabidopsis reduced the anthocyanin accumulation level. Transient promoter activation assays revealed that CsMYB3 could repress the activation capacity of the complex formed by CsRuby1/CsbHLH1 for the anthocyanin biosynthetic genes. Moreover, CsMYB3 could be transcriptionally activated by CsRuby1 via promoter binding, thus forming an 'activator-and-repressor' loop to regulate anthocyanin biosynthesis in citrus. This study shows that CsMYB3 plays a repressor role in the regulation of anthocyanin biosynthesis and proposes an 'activator-and-repressor' loop model constituted by CsRuby1 and CsMYB3 in the regulation of anthocyanin biosynthesis in citrus.

Retrotransposon promoter of Ruby1 controls both light- and cold-induced accumulation of anthocyanins in blood orange.

Blood orange is generally recognized to accumulate anthocyanins in its fruit pulp in a cold-inducible manner. We observed that the fruit peel of blood orange can also accumulate anthocyanins under ample light conditions. Interestingly, purple pummelo can accumulate anthocyanins only in its fruit peel but not in its pulp. The mechanism underlying the tissue specificity of anthocyanin accumulation in citrus is unknown. Here, we show that the active promoter of Ruby1, a key activator of anthocyanin biosynthesis, is also light inducible in addition to its already known cold inducibility in blood orange. Electrophoretic mobility shift assays and transient expression assays showed that HY5 positively regulated the transcription of Ruby1 by binding to the G-box motif (CACGTC). The tissue specificity of anthocyanin accumulation in the peel of purple pummelo may be due to the lack of a low temperature responsive element and a MYC binding site, which were shown to be involved in cold inducibility of CsRuby1 in blood orange by insertion of a long terminal repeat type retrotransposon in the promoter. These results bring new insights into the regulatory mechanism of anthocyanin biosynthesis in response to environmental stimuli and provide cis-elements for genetic improvement of anthocyanin-stable fruits rich in antioxidant metabolites.

Subfunctionalization of the Ruby2-Ruby1 gene cluster during the domestication of citrus.

The evolution of fruit colour in plants is intriguing. Citrus fruit has repeatedly gained or lost the ability to synthesize anthocyanins. Chinese box orange, a primitive citrus, can accumulate anthocyanins both in its fruits and its leaves. Wild citrus can accumulate anthocyanins in its leaves. In contrast, most cultivated citrus have lost the ability to accumulate anthocyanins. We characterized a novel MYB regulatory gene, Ruby2, which is adjacent to Ruby1, a known anthocyanin activator of citrus. Different Ruby2 alleles can have opposite effects on the regulation of anthocyanin biosynthesis. AbRuby2Full encodes an anthocyanin activator that mainly functions in the pigmented leaves of Chinese box orange. CgRuby2Short was identified in purple pummelo and encodes an anthocyanin repressor. CgRuby2Short has lost the ability to activate anthocyanin biosynthesis. However, it retains the ability to interact with the same partner, CgbHLH1, as CgRuby1, thus acting as a passive competitor in the regulatory complex. Further investigation in different citrus species indicated that the Ruby2-Ruby1 cluster exhibits subfunctionalization among primitive, wild and cultivated citrus. Our study elucidates the regulatory mechanism and evolutionary history of the Ruby2-Ruby1 cluster in citrus, which are unique and different from that found in Arabidopsis, grape or petunia.