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
Project description:Cajal bodies (CBs) are membraneless organelles whose mechanism of formation is still not fully understood. Many proteins contribute to the formation of CBs, including Nopp140, WRAP53, and coilin. SUMOylation, a post-translational modification, of coilin involves multiple different lysine residues. Additionally, coilin is also found in the nucleoplasm where its role is still being understood. Here, we demonstrate a novel mechanism examining the interaction changes of coilin when its SUMOylation is disrupted. We look at both global SUMOylation inhibition and its effect on CB formation, as well as targeted SUMOylation inhibition of coilin itself in cells by developing a coilin SUMO mutant. We found upon two types of global SUMOylation inhibition, as well as upon transfection of the coilin SUMO mutant, that CBs increased in number and decreased in size. Additionally, we saw via coimmunoprecipitation the coilin SUMO mutant has altered interaction with Nopp140. This demonstrates increased mechanistic ties to CB formation and SUMOylation. Furthermore, RNA sequencing and small RNA sequencing on a primary cell line with few CBs showed that coilin impacts the response to lipopolysaccharide-induced stress. Collectively, our results identify SUMOylation as a novel mechanism driving CB formation and highlights a positive role for human coilin in innate immunity.
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.
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. 5 biological replicates from P19 and 2 biological replicates from HeLa cells after UV-crosslinking. Negative control samples prepared from GFP-NLS fusion protein are stored uder accession E-MTAB-747.
Project description:Cajal bodies (CBs) are membraneless organelles whose mechanism of formation is still not fully understood. Many proteins contribute to the formation of CBs, including Nopp140, WRAP53, and coilin. SUMOylation, a post-translational modification, of coilin involves multiple different lysine residues. Additionally, coilin is also found in the nucleoplasm where its role is still being understood. Here, we demonstrate a novel mechanism examining the interaction changes of coilin when its SUMOylation is disrupted. We look at both global SUMOylation inhibition and its effect on CB formation, as well as targeted SUMOylation inhibition of coilin itself in cells by developing a coilin SUMO mutant. We found upon two types of global SUMOylation inhibition, as well as upon transfection of the coilin SUMO mutant, that CBs increased in number and decreased in size. Additionally, we saw via coimmunoprecipitation the coilin SUMO mutant has altered interaction with Nopp140. This demonstrates increased mechanistic ties to CB formation and SUMOylation. Furthermore, RNA sequencing and small RNA sequencing on a primary cell line with few CBs showed that coilin impacts the response to lipopolysaccharide-induced stress. Collectively, our results identify SUMOylation as a novel mechanism driving CB formation and highlights a positive role for human coilin in innate immunity.
Project description:Coilin is a nuclear protein known for its roles in assembling the Cajal Body and participating in the biogenesis of ribonucleoproteins. We have uncovered novel functions for coilin in promoting microRNA biogenesis and the phosphorylation/SUMOylation of various proteins, through which we have observed a role for coilin in responding to stress signals. Coilin has also been found to participate in the stress response in plants through the regulated expression of immunity genes and activation of defense mechanisms. In this study, we further investigated this role of coilin in regulating the immune response by applying transcriptomic analyses to examine the response to lipopolysaccharide-mediated stress in coilin-depleted human cells and coilin mutant zebrafish embryos. Our results indicate a vital function for coilin in regulating the expression of immunity-related genes and establish a conserved role for vertebrate coilin in the promotion of the innate immune response across humans and zebrafish.
Project description:Coilin iCLIP data revealed 42 novel human snoRNAs of intronic origin. To validate their expression and estimate abundance of novel and annotated snoRNAs, we performed RNA-seq on polyA- and rRNA-depleted RNA isolated from HeLa cells. Results show that expression of novel snoRNAs is comparable to the previously annotated snoRNAs. 1 replicate of RNA depleted of polyA and ribosomal RNA.
Project description:Coilin iCLIP data revealed 42 novel human snoRNAs of intronic origin. To validate their expression and estimate abundance of novel and annotated snoRNAs, we performed RNA-seq on polyA- and rRNA-depleted RNA isolated from HeLa cells. Results show that expression of novel snoRNAs is comparable to the previously annotated snoRNAs.