The MYB transcription factors and plant hormone ABA have been suggested to play a role in fruit anthocyanin biosynthesis, but supporting genetic evidence has been lacking in sweet cherry. The present study describes the first functional characterization of an R2R3-MYB transcription factor, PacMYBA, from red-colored sweet cherry cv. Hong Deng (Prunus avium L.). Transient promoter assays demonstrated that PacMYBA physically interacted with several anthocyanin-related basic helix-loop-helix (bHLH) transcription factors to activate the promoters of PacDFR, PacANS and PacUFGT, which are thought to be involved in anthocyanin biosynthesis. Furthermore, the immature seeds of transgenic Arabidopsis plants overexpressing PacMYBA exhibited ectopic pigmentation. Silencing of PacMYBA, using a Tobacco rattle virus (TRV)-induced gene silencing technique, resulted in sweet cherry fruit that lacked red pigment. ABA treatment significantly induced anthocyanin accumulation, while treatment with the ABA biosynthesis inhibitor nordihydroguaiaretic acid (NDGA) blocked anthocyanin production. PacMYBA expression peaked after 2 h of pre-incubation in ABA and was 15.2-fold higher than that of sweet cherries treated with NDGA. The colorless phenotype was also observed in the fruits silenced in PacNCED1, which encodes a key enzyme in the ABA biosynthesis pathway. The endogenous ABA content as well as the transcript levels of six structural genes and PacMYBA in PacNCED1-RNAi (RNA interference) fruit were significantly lower than in the TRV vector control fruit. These results suggest that PacMYBA plays an important role in ABA-regulated anthocyanin biosynthesis and ABA is a signal molecule that promotes red-colored sweet cherry fruit accumulating anthocyanin.

Arabidopsis Argonaute10 (AGO10) specifically sequesters miR165 and miR166 and antagonizes their activity, thus regulating shoot apical meristem (SAM) development. However, where and when this sequestration acts is currently unclear. We show here that AGO10 represses miR165/166 activity in the embryo proper during early embryogenesis, through the apical and central regions of mature embryos, and eventually in the entire adaxial domain and vasculature of the cotyledons and leaf primordia. These locations are essentially identical to regions expressing PHABULOSA and REVOLUTA, mRNA targets of miR165/166. The Arabidopsis genome contains nine MIR165/166 genes. Sequestration of miR165/166 by the MIR165b, MIR166a, MIR166b, and MIR166g promoters efficiently rescues the SAM defect in ago10 mutants. Comparison of the expression patterns of AGO10 and the four MIR165/166 members suggests that AGO10 quenches the non-cell-autonomous activity of any miR165/166 that moves into AGO10-expressing niches. Thus, this study provides insight into how the spatiotemporal regulation of AGO10-miR165/166 activity affects SAM development.

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Argonaute (AGO) proteins recruit 21-24-nucleotide (nt) small RNAs (sRNAs) to constitute RNA-induced silencing complexes (RISCs) to regulate gene expression at transcriptional or posttranscriptional levels(1-3). Arabidopsis encodes nine functional AGO proteins. These proteins are classified into three clusters, AGO1/5/10, AGO2/3/7 and AGO4/6/9, based on their sequence similarity, functional redundancy, as well as species and features of AGO-bound sRNAs(4-7). Although most Arabidopsis AGO proteins have been studied well, AGO3-bound sRNAs and their basic function remain unknown. Here we observed that AGO3 could not complement the signature function of AGO2, the closest genetic paralog of AGO3, in host antiviral defence. We also found, surprisingly, that AGO3 predominantly bound 24-nt sRNAs with 5'-terminal adenine. The spectrum of AGO3-associated sRNAs was different from those bound to AGO2, further indicating their functional divergence. By contrast, approximately 30% of AGO3-bound 24-nt sRNAs overlapped with those bound to AGO4, and over 60% of AGO3-associated 24-nt sRNA-enriched loci were identical to those of AGO4. Moreover, the redundancy of AGO3- and AGO4-bound sRNAs is much more than that of AGO6- and AGO4-recruited sRNAs. In addition, expression of AGO3 driven by the AGO4 promoter partially complemented AGO4 function and rescued a DNA methylation defect in the ago4-1 background. Together, our results indicated that AGO3, similarly to AGO4, is a component in the epigenetic pathway.

MicroRNA (miRNA) is processed from primary transcripts with hairpin structures (pri-miRNAs) by microprocessors in the nucleus. How cytoplasmic-borne microprocessor components are transported into the nucleus to fulfill their functions remains poorly understood. Here, we report KETCH1 (karyopherin enabling the transport of the cytoplasmic HYL1) as a partner of hyponastic leaves 1 (HYL1) protein, a core component of microprocessor in and functional counterpart of DGCR8/Pasha in animals. Null mutation of is embryonic-lethal, whereas knockdown mutation of caused morphological defects, reminiscent of mutants in the miRNA pathway. knockdown mutation also substantially reduced miRNA accumulation, but did not alter nuclear-cytoplasmic shuttling of miRNAs. Rather, the mutation significantly reduced nuclear portion of HYL1 protein and correspondingly compromised the pri-miRNA processing in the nucleus. We propose that KETCH1 transports HYL1 from the cytoplasm to the nucleus to constitute functional microprocessor in This study provides insight into the largely unknown nuclear-cytoplasmic trafficking process of miRNA biogenesis components through eukaryotes.

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Diverse RNA transcripts acting as competing endogenous RNAs (ceRNAs) can co-regulate each other's expression by competing for shared microRNAs. CCR2 protein, the receptor for CCL2, is implicated in cancer progression. However, we found that a higher mRNA level is remarkably associated with prolonged survival of breast cancer patients. These conflicting results prompted us to study the non-coding function of mRNA. We found that the 3' untranslated region (UTR) inhibited MDA-MB-231 and MCF-7 cell metastasis by repressing epithelial-mesenchymal transition (EMT), and suppressed breast cancer metastasis Mechanistically, the 3'UTR modulated the expression of the RhoGAP protein STARD13 via acting as a STARD13 ceRNA in a microRNA-dependent and protein coding-independent manner. The 3'UTR blocked the activation of RhoA-ROCK1 pathway, which is the downstream effector of STARD13, and thus decreased the phosphorylation level of myosin light chain 2 (MLC2) and formation of F-actin. Additionally, the function of the 3'UTR was dependent on STARD13 expression. In conclusion, our results confirmed that the 3'UTR acts as a metastasis suppressor by acting as a ceRNA for STARD13 and thus inhibiting RhoA-ROCK1-MLC-F-actin pathway in breast cancer cells.This article has an associated First Person interview with the first author of the paper.

ObjectiveTo investigate the therapeutic effect and survival outcome using nomogram by incorporating significant inflammatory markers in patients with thoracic esophageal squamous cell carcinoma (ESCC) who received chemoradiotherapy (CRT) or single radiotherapy (RT).MethodA total of 266 patients diagnosed with thoracic ESCC receiving standard curative RT only or concurrent CRT were retrospectively analysed. The patients were grouped for statistical analysis depending on the median values of neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and C-reactive protein/albumin (CRP/Alb) ratio. The therapeutic effect was analysed by univariate and multivariate logistic analyses. The survival prognosis was estimated by univariate and multivariate Cox analyses. At last, the nomogram was developed by incorporating the significant inflammatory markers and clinicopathological parameters, and the predictive value was verified by calibration curve, concordance index (C-index) and decision curve.ResultsThe treatment responses were highly associated with clinical stage, tumor location, NLR, PLR and CRP/Alb ratio (all P<0.05) by univariate logistic analysis. However, in the multivariate logistic analysis, the results showed that only CRP/Alb ratio (P=0.000) and TNM stage (P=0.008) were independent risk parameters for tumour response. In addition, NLR, PLR, CRP/Alb ratio, age and TNM stage were significantly associated with OS by the univariate Cox analysis (all P<0.05). Furthermore, the multivariate Cox analysis showed that only CRP/Alb ratio (P=0.000), TNM stage (P=0.000) and age (P=0.001) were considered independent prognostic factors for OS. Finally, the calibration curves of nomogram were highly consistent with actual observation for the therapeutic effect and prognosis, and the decision curve analysis showed more potential of clinical benefit of the nomogram compared with TNM staging system.ConclusionThis research found that nomogram-integrated CRP/Alb ratio was promising as a predictive model for the therapeutic effect and survival outcome in patients with thoracic ESCC receiving CRT or single RT.