Browsing by Author "Shen, Yun"
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Item The Arabidopsis RNA Binding Protein with K Homology Motifs, SHINY1, Interacts with the C-terminal Domain Phosphatase-like 1 (CPL1) to Repress Stress-Inducible Gene Expression(2013) Jiang, Jiafu; Wang, Bangshing; Shen, Yun; Wang, Hui; Feng, Qing; Shi, HuazhongThe phosphorylation state of the C-terminal domain (CTD) of the RNA polymerase II plays crucial roles in transcription and mRNA processing. Previous studies showed that the plant CTD phosphatase-like 1 (CPL1) dephosphorylates Ser-5-specific CTD and regulates abiotic stress response in Arabidopsis. Here, we report the identification of a K-homology domain-containing protein named SHINY1 (SHI1) that interacts with CPL1 to modulate gene expression. The shi1 mutant was isolated from a forward genetic screening for mutants showing elevated expression of the luciferase reporter gene driven by a salt-inducible promoter. The shi1 mutant is more sensitive to cold treatment during vegetative growth and insensitive to abscisic acid in seed germination, resembling the phenotypes of shi4 that is allelic to the cpl1 mutant. Both SHI1 and SHI4/CPL1 are nuclear-localized proteins. SHI1 interacts with SHI4/CPL1 in vitro and in vivo. Loss-of-function mutations in shi1 and shi4 resulted in similar changes in the expression of some stress-inducible genes. Moreover, both shi1 and shi4 mutants display higher mRNA capping efficiency and altered polyadenylation site selection for some of the stress-inducible genes, when compared with wild type. We propose that the SHI1-SHI4/CPL1 complex inhibits transcription by preventing mRNA capping and transition from transcription initiation to elongation.Item The DEAD-box RNA helicase SHI2 functions in repression of salt-inducible genes and regulation of cold-inducible gene splicing(2020) Wang, Bangshing; Chai, Haoxi; Zhong, Yingli; Shen, Yun; Yang, Wannian; Chen, Junping; Xin, Zhanguo; Shi, HuazhongGene regulation is central for growth, development, and adaptation to environmental changes in all living organisms. Many genes are induced by environmental cues, and the expression of these inducible genes is often repressed under normal conditions. Here, we show that the SHINY2 (SHI2) gene is important for repressing salt-inducible genes and also plays a role in cold response. The shi2 mutant displayed hypersensitivity to cold, abscisic acid (ABA), and LiCl. Map-based cloning demonstrates that SHI2 encodes a DEAD- (Asp-Glu-Ala-Asp) box RNA helicase with similarity to a yeast splicing factor. Transcriptomic analysis of the shi2 mutant in response to cold revealed that the shi2 mutation decreased the number of cold-responsive genes and the magnitude of their response, and resulted in the mis-splicing of some cold-responsive genes. Under salt stress, however, the shi2 mutation increased the number of salt-responsive genes but had a negligible effect on mRNA splicing. Our results suggest that SHI2 is a component in a ready-for-transcription repressor complex important for gene repression under normal conditions, and for gene activation and transcription under stress conditions. In addition, SHI2 also serves as a splicing factor required for proper splicing of cold-responsive genes and affects 5' capping and polyadenylation site selection.Item Map-based cloning and functional characterization of Arabidopsis LHR1 and SHI7 genes(2014-05) Shen, Yun; Shi, Huazhong; Zhang, Hong; Pare, Paul; Xie, ZhixinIn an effort to identify novel genetic components that involve in the gene regulation and signaling pathways of plant abiotic stress response and tolerance, two forward genetic mutant screening systems were established by utilizing the stress-inducible promoters of AtHSP18.2 and AtSOT12 genes and the LUC reporter gene. A number of mutants displaying altered luciferase activity in response to abiotic stresses were isolated. In this study, two mutants named lhr1 and shi7 were subjected to map-based cloning and further molecular and physiological characterizations. The lhr1 mutant contains a single recessive mutation which leads to reduced expression of the LUC reporter gene under both normal and heat stress conditions comparing with wild type. Map-based cloning of the lhr1 mutation identified a single nucleotide missense change in a gene encoding a polyamine uptake transporter. LHR1 protein contains 12 transmembrane domains and belongs to an amino acid permease family. The lhr1 mutant shows tolerance to paraquat and cycloheximide, hypersensitivity to ABA and NaCl in seed germination, and altered expression of several abotic stress-responsive genes. Further study showed LHR1 is engaged in the transport of extracellular paraquat and polyamines into cytoplasm. The shi7 mutant has a single recessive mutation causing higher LUC expression after both NaCl and sorbitol treatments when compared with wild type. A single nucleotide missense mutation in a gene encoding an ARF-GAP DOMAIN protein was identified in the shi7 mutant by Map-based cloning. The SHI7 protein contains 828 amino acid residues and consists of four conserved domains including an ARF-GAP domain. The shi7 mutant displays root hair biogenesis deficiency in newly formed maturation zone of root, but shows no apparent morphological phenotypes under tested stress conditions. Both genetic and molecular complementation confirmed that the mutant phenotype of shi7 is caused by the mutation in the SHI7 gene. Both LHR1 and SHI7 genes play important roles in abiotic stress response in Arabidopsis. The molecular mechanisms by which these two genes modulate stress-responsive gene expression were further studied, and the direction of future research were also discussed.Item A nematode sterol C4α-methyltransferase catalyzes a new methylation reaction responsible for sterol diversity(2020) Zhou, Wenxu; Fisher, Paxtyn M.; Vanderloop, Boden H.; Shen, Yun; Shi, Huazhong; Maldonado, Adrian J.; Leaver, David J.; Nes, W. DavidPrimitive sterol evolution plays an important role in fossil record interpretation and offers potential therapeutic avenues for human disease resulting from nematode infections. Recognizing that C4-methyl stenol products [8(14)-lophenol] can be synthesized in bacteria while C4-methyl stanol products (dinosterol) can be synthesized in dinoflagellates and preserved as sterane biomarkers in ancient sedimentary rock is key to eukaryotic sterol evolution. In this regard, nematodes have been proposed to convert dietary cholesterol to 8(14)-lophenol by a secondary metabolism pathway that could involve sterol C4 methylation analogous to the C2 methylation of hopanoids (radicle-type mechanism) or C24 methylation of sterols (carbocation-type mechanism). Here, we characterized dichotomous cholesterol metabolic pathways in Caenorhabditis elegans that generate 3-oxo sterol intermediates in separate paths to lophanol (4-methyl stanol) and 8(14)-lophenol (4-methyl stenol). We uncovered alternate C3-sterol oxidation and Δ7 desaturation steps that regulate sterol flux from which branching metabolite networks arise, while lophanol/8(14)-lophenol formation is shown to be dependent on a sterol C4α-methyltransferse (4-SMT) that requires 3-oxo sterol substrates and catalyzes a newly discovered 3-keto-enol tautomerism mechanism linked to S-adenosyl-l-methionine-dependent methylation. Alignment-specific substrate-binding domains similarly conserved in 4-SMT and 24-SMT enzymes, despite minimal amino acid sequence identity, suggests divergence from a common, primordial ancestor in the evolution of methyl sterols. The combination of these results provides evolutionary leads to sterol diversity and points to cryptic C4-methyl steroidogenic pathways of targeted convergence that mediate lineage-specific adaptations.