Alternative splicing factor or splicing factor-2 plays a key role in intron retention of the endoglin gene during endothelial senescence

Alternative splicing involving intron retention plays a key role in the regulation of gene expression. We previously reported that the alternatively spliced short isoform of endoglin (S-endoglin) is induced during the aging or senescence of endothelial cells by a mechanism of intron retention. In this work, we demonstrate that the alternative splicing factor or splicing factor-2 (ASF/SF2) is involved in the synthesis of endoglin. Overexpression of ASF/SF2 in endothelial cells switched the balance between the two endoglin isoforms, favoring the synthesis of S-endoglin. Using a minigene reporter vector and RNA immunoprecipitation experiments, it was shown that ASF/SF2 interacts with the nucleotide sequence of the endoglin minigene, suggesting the direct involvement of ASF/SF2. Accordingly, the sequence recognized by ASF/SF2 in the endoglin gene was identified inside the retained intron near the consensus branch point. Finally, the ASF/SF2 subcellular localization during endothelial senescence showed a preferential scattered distribution throughout the cytoplasm, where it interferes with the activity of the minor spliceosome, leading to an increased expression of S-endoglin mRNA. In summary, we report for the first time the molecular mechanisms by which ASF/SF2 regulates the alternative splicing of endoglin in senescent endothelial cells, as well as the involvement of ASF/SF2 in the minor spliceosome.     

可变剪接调控植物开花的作用机制进展

开花是植物生长发育的关键转折,与种子生产和作物产量密切相关。开花转变受到复杂的基因网络调控,许多开花相关基因通过可变剪接产生多种转录本,调控开花时间。文中从多个角度系统地综述了可变剪接调控植物开花的分子机制,并对将来的研究进行了展望。     

Identification of a prognostic alternative splicing signature in oral squamous cell carcinoma

Alternative splicing (AS) is critically associated with tumorigenesis and patient's prognosis. Here, we systematically analyzed survival-associated AS signatures in oral squamous cell carcinoma (OSCC) and evaluated their prognostic predictive values. Survival-related AS events were identified by univariate and multivariate Cox regression analyses using OSCC data from the TCGA head neck squamous cell carcinoma data set. The Percent Spliced In calculated by SpliceSeq from 0 to 1 was used to quantify seven types of AS events. A predictive model based on AS events was constructed by least absolute shrinkage and selection operator Cox regression assay and further validated using a training-testing cohort design. Patient survival was estimated using the Kaplan–Meier method and compared with Log-rank test. The receiver operating characteristics curve area under the curves was used to evaluate the predictive abilities of these predictive models. Furthermore, gene–gene interaction networks and the splicing factors (SFs)-AS regulatory network was generated by Cytoscape. A total of 825 survival-related AS events within 719 genes were identified in OSCC samples. The integrative predictive model was better at predicting outcomes of patients as compared to those models built with the individual AS event. The predictive model based on three AS-related genes also effectively predicted patients’ survival. Moreover, seven survival-related SFs were detected in OSCC including RBM4, HNRNPD, and HNRNPC, which have been linked to tumorigenesis. The SF-AS network revealed a significant correlation between survival-related AS genes and these SFs. Our findings revealed a systemic portrait of survival-associated AS events and the splicing network in OSCC, suggesting that AS events might serve as novel prognostic biomarkers and therapeutic targets for OSCC.     

Combined biochemical and electron microscopic analyses reveal the architecture of the mammalian U2 snRNP.

The 17S U2 small nuclear ribonucleoprotein particle (snRNP) represents the active form of U2 snRNP that binds to the pre-mRNA during spliceosome assembly. This particle forms by sequential interactions of splicing factors SF3b and SF3a with the 12S U2 snRNP. We have purified SF3b and the 15S U2 snRNP, an intermediate in the assembly pathway, from HeLa cell nuclear extracts and show that SF3b consists of four subunits of 49, 130, 145, and 155 kD. Biochemical analysis indicates that both SF3b and the 12S U2 snRNP are required for the incorporation of SF3a into the 17S U2 snRNP. Nuclease protection studies demonstrate interactions of SF3b with the 5' half of U2 small nuclear RNA, whereas SF3a associates with the 3' portion of the U2 snRNP and possibly also interacts with SF3b. Electron microscopy of the 15S U2 snRNP shows that it consists of two domains in which the characteristic features of isolated SF3b and the 12S U2 snRNP are conserved. Comparison to the two-domain structure of the 17S U2 snRNP corroborates the biochemical results in that binding of SF3a contributes to an increase in size of the 12S U2 domain and possibly induces a structural change in the SF3b domain.     

Isolation of Z-3-Hexenal in Tea Leaves,Thea sinensis,and Synthesis Thereof

Z-3-Hexenal, a precursor in the biosynthesis of leaf alcohol and leaf aldehyde, was first isolated from fresh tea leaves, Thea sinensis and its structure was confirmed by unequivocal synthetic evidence.     

可变剪接在植物免疫中的研究进展

可变剪接是生物重要的转录后修饰过程,是转录组和蛋白组多样性的重要来源.可变剪接参与了植物众多生理过程,包括植物昼夜节律、生长发育等,在植物响应生物和非生物胁迫过程中尤为普遍.近年来,可变剪接被认为是植物抵御病原菌侵染的重要调控机制.本文综述了可变剪接在植物免疫各个层面的调控作用,包括调节重要免疫受体、R基因、激素信号路...     

Comprehensively analysis of splicing factors to construct prognosis prediction classifier in prostate cancer

Splicing factors (SFs) are proteins that control the alternative splicing (AS) of RNAs, which have been recognized as new cancer hallmarks. Their dysregulation has been found to be involved in many biological processes of cancer, such as carcinogenesis, proliferation, metastasis and senescence. Dysregulation of SFs has been demonstrated to contribute to the progression of prostate cancer (PCa). However, a comprehensive analysis of the prognosis value of SFs in PCa is limited. In this work, we systematically analysed 393 SFs to deeply characterize the expression patterns, clinical relevance and biological functions of SFs in PCa. We identified 53 survival-related SFs that can stratify PCa into two de nove molecular subtypes with distinct mRNA expression and AS-event expression patterns and displayed significant differences in pathway activity and clinical outcomes. An SF-based classifier was established using LASSO-COX regression with six key SFs (BCAS1, LSM3, DHX16, NOVA2, RBM47 and SNRPN), which showed promising prognosis-prediction performance with a receiver operating characteristic (ROC) >0.700 in both the training and testing datasets, as well as in three external PCa cohorts (DKFZ, GSE70769 and GSE21035). CRISPR/CAS9 screening data and cell-level functional analysis suggested that LSM3 and DHX16 are essential factors for the proliferation and cell cycle progression in PCa cells. This study proposes that SFs and AS events are potential multidimensional biomarkers for the diagnosis, prognosis and treatment of PCa.     

选择性剪接在低氧应答中的调控作用

细胞为适应低氧环境,其相关基因的表达方式发生了改变,其中选择性剪接在低氧应答调控过程中起到了重要的作用。低氧诱导因子介导的低氧应答信号通路在机体适应低氧环境过程中起到了十分重要的作用,低氧诱导因子剪接体通过此通路调控红细胞生成、血管生成、糖酵解等过程。而抑制性PAS蛋白质、脯氨酸羟化酶、促血管生长因子、芳香羟受体核转运蛋白剪接体则通过其它通路进行调控。选择性剪接不仅在低氧应答中起重要作用,而且与阿尔茨海默病、动脉粥样硬化、癌症等常见人类疾病相关。     

Alternative mRNA processing increases the complexity of microRNA-based gene regulation in Arabidopsis

During trans-splicing of discontinuous organellar introns, independently transcribed coding sequences are joined together to generate a continuous mRNA. The chloroplast psaA gene from Chlamydomonas reinhardtii encoding the P(700) core protein of photosystem I (PSI) is split into three exons and two group IIB introns, which are both spliced in trans. Using forward genetics, we isolated a novel PSI mutant, raa4, with a defect in trans-splicing of the first intron. Complementation analysis identified the affected gene encoding the 112.4 kDa Raa4 protein, which shares no strong sequence identity with other known proteins. The chloroplast localization of the protein was confirmed by confocal fluorescence microscopy, using a GFP-tagged Raa4 fusion protein. RNA-binding studies showed that Raa4 binds specifically to domains D2 and D3, but not to other conserved domains of the tripartite group II intron. Raa4 may play a role in stabilizing folding intermediates or functionally active structures of the split intron RNA.     

利用内蛋白子剪切功能一步纯化重组人神经营养因子-3

将人神经营养因子 - 3(h NT3)基因插入含内蛋白子 -几丁质结合区 (Intein- CBD)片段的质粒p TXB1的多克隆位点 ,构建成重组子 p TXB- h NT3,随后转化入 E.coli 2 566并进行融合表达 .表达产物包涵体经 8mol/ L脲变性 ,并在 GSH,GSSG存在下复性 .复性后的融合蛋白经几丁质珠亲和柱吸附 .待洗涤杂蛋白后 ,加入 50 mmol/ L DTT在 4℃或 2 5℃进行剪切反应 48h,再用缓冲液洗脱 ,即得 h NT3.SDS- PAGE分析表明 ,h NT3达电泳纯 .其分子量约为 1 4 k D     

Transport of galectin-3 between the nucleus and cytoplasm. II. Identification of the signal for nuclear export

Galectin-3, a factor involved in the splicing of pre-mRNA, shuttles between the nucleus and the cytoplasm. Previous studies have shown that incubation of fibroblasts with leptomycin B resulted in the accumulation of galectin-3 in the nucleus, suggesting that the export of galectin-3 from the nucleus may be mediated by the CRM1 receptor. A candidate nuclear export signal fitting the consensus sequence recognized by CRM1 can be found between residues 240 and 255 of the murine galectin-3 sequence. This sequence was engineered into the pRev(1.4) reporter system, in which candidate sequences can be tested for nuclear export activity in terms of counteracting the nuclear localization signal present in the Rev(1.4) protein. Rev(1.4)-galectin-3(240-255) exhibited nuclear export activity that was sensitive to inhibition by leptomycin B. Site-directed mutagenesis of Leu247 and Ile249 in the galectin-3 nuclear export signal decreased nuclear export activity, consistent with the notion that these two positions correspond to the critical residues identified in the nuclear export signal of the cAMP-dependent protein kinase inhibitor. The nuclear export signal activity was also analyzed in the context of a full-length galectin-3 fusion protein; galectin-3(1-263; L247A) showed more nuclear localization than wild-type, implicating Leu247 as critical to the function of the nuclear export signal. These results indicate that residues 240-255 of the galectin-3 polypeptide contain a leucine-rich nuclear export signal that overlaps with the region (residues 252-258) identified as important for nuclear localization.     

前肽缺失vWF促进细胞分泌蛋白质剪接的L303E／F309S突变体凝血第八因子

该文旨在探索前肽缺失von Willebrand子（vWF-△Pro）对蛋白质剪接的L303E／F309S突变体凝血第八因子（FVIII）分泌的影响。将vWF-△Pro基因与蛋白内含子融合的F聊重链和轻链基因共转HEK293胞。结果显示,转vWF－△Pro细胞的剪接蛋白FVIll分泌量和活性分别为（196±27）ng／mL和（1．39±0-31）IU／mL,明显高于对照细胞的（116±24）ng／mL和（0．91±0．18）IU／mL。表明vWF－△Pro可提高剪接的L303E／F309S突变体FVⅢ蛋白分泌量和活性。