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Arabidopsis COILIN orchestrates transcriptional as well as post-transcriptional regulation to combat pathogens

ABSTRACT: The objective of the study was to understand the role of Coilin in plant pathogen interactions.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE182639 | GEO | 2022/06/30

REPOSITORIES: GEO

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Arabidopsis COILIN orchestrates transcriptional as well as post-transcriptional regulation to combat pathogens

Project description:Arabidopsis COILIN orchestrates transcriptional as well as post-transcriptional regulation to combat pathogens

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iCLIP mapping of coilin-RNA interactions in HeLa and P19 cells

Project description:Our ChIP resuls suggested that coilin association with U3, snRNA and histone genes might be dependent on coilin-RNA interaction. We used iCLIP of coilin-GFP expressed in HeLa and P19 cell lines at endogenous levels to identify coilin RNA targets and investigate RNA-binding specificity. P19 cells expressing GFP fused to a nuclear localization signal (GFP-NLS) was used as a negative control. iCLIP results revealed that coilin binds several classes of ncRNA including snRNAs, U3 snoRNA and scaRNAs. Interestlignly the majority of coilin targets were intronic snoRNAs, suggesting a novel role of CBs in snoRNA biogenesis.

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Coilin ChIP-seq in cycling HeLa cells

Project description:To investigate location of Cajal bodies (CB) inside the cell nucleus, we created a stable cell line expressing the GFP version of CB-marker protein coilin at endogenous levels and performed ChIP-seq with anti-EGFP antibody. ChIP-seq revealed close association of CBs wih U3 genes, snRNA genes and histone genes in histone cluster 1 and 2. 1 biological replicate of coilin ChIP-seq and input sample

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Coilin ChIP-seq in cycling HeLa cells

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Phosphorylation detection of protein DGCR8 between between control and coilin knockdown samples

Project description:The project is for identification and quantification of phosphorylated peptides of protein DGCR8 between control and coilin knockdown samples.

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Identification of coilin interactors reveals coordinated control of Cajal body number and substructure

Project description:The cell nucleus contains distinct biomolecular condensates that form at specific genetic loci, organize chromosomes in 3D space, and regulate RNA processing. Among these, Cajal bodies (CBs) require key “scaffolding” proteins for their assembly, which is not fully understood. Here we employ proximity biotinylation, mass spectrometry, and functional screening to comprehensively identify and test functions of CB components. We document 144 protein interactors of coilin, of which 70 were newly detected, and establish 25 players needed for CB assembly and/or maintenance. Surprisingly, depletion of nine coilin interactors – mostly constituents of the 60S ribosome (RPLs) – increased CB number and caused subdomains defined by coilin and the Survival of Motor Neuron protein (SMN) to merge. These phenotypes were traceable to altered nuclear levels of dimethylarginine. Our data implicate RPL24 and other players in the regulation of CBs by modulating post-translational modifications. Moreover, the prevalence of transcription factors among the identified components highlights roles for gene activity in CB assembly and nuclear positioning.

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Coilin and Pol II ChIP-seq in HeLa cells after RPL14 and RPL24 depletion

Project description:To investigate location of Cajal bodies (CB) inside the cell nucleus after RPL depletion, we performed ChIP-seq with an anti-Coilin antibody after siRNA knockdown of RPL14 and RPL24. ChIP-seq revealed a loss of association of CBs wih snRNA genes and histone genes in histone cluster 1 and 2 upon RPL KD. We also performed Pol 2 ChIP-seq to look at transcription of these gene loci upon RPL knockdown and found decreased Pol II binding along genes.

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A coilin mutant provides a sensitized system for identifying modifiers of alternative splicing and posttranscriptional gene silencing in Arabidopsis thaliana

Project description:Coilin is a scaffold protein essential for the structural integrity of Cajal Bodies, which are non-membranous nuclear organelles that are thought to facilitate assembly and maturation of nuclear RNPs, including spliceosomal snRNPs. To investigate further coilin’s functions in plant cells, and to identify proteins that may functionally interact with coilin, we performed a genetic suppressor screen in Arabidopsis thaliana using a coilin (coi) mutant displaying altered splicing of a GFP pre-mRNA. The modified splicing pattern results in a ‘hyper-GFP’ phenotype in young coi seedlings relative to the intermediate level of GFP in wild-type seedlings. Additionally, in newly emerging leaves of older coi seedlings, the GFP gene frequently undergoes abrupt siRNA-associated posttranscriptional gene silencing that persists during growth. In the suppressor screen, we searched for mutations that subdue one or both of these GFP phenotypes and identified several understudied factors in plants: WRAP53, a putative Cajal body protein; SMU2, a predicted splicing-related factor; and ZC3HC1, an uncharacterized zinc finger protein. All three mutations return the hyper-GFP phenotype of the coi mutant to approximately the intermediate wild-type level. The zc3hc1 mutations in particular induce premature and more extensive posttranscriptional gene silencing similar to mutations in SOP1 and DCL4, which are known modifiers of posttranscriptional gene silencing. Candidate coilin-interacting proteins identified by immunoprecipitation-mass spectrometry include many splicing-related factors, nucleolar proteins, and mRNA export factors. Our results demonstrate the usefulness of the coi mutant to identify new modifiers of alternative splicing and posttranscriptional gene silencing, and suggest diverse roles for coilin in plant cells.

2023-07-27 | PXD027146 | Pride

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