Individual-nucleotide resolution CLIP and high-throughput sequencing to map hnRNP L-RNA interactions in HeLa cells
ABSTRACT: Individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) combined with high-throughput sequencing was used to generate a transcriptome-wide binding map of hnRNP L. Supplementary file GSE37560_hnRNPL_crosslink_site.bed includes filtered crosslink sites of hnRNPL: combining data from all 3 experiments. 3 biological replicates of hnRNP L-specific and control (Flag) co-immunoprecipitated RNA after UV-crosslinking in HeLa cells
Project description:Precise control of mRNA decay is fundamental for robust yet not exaggerated inflammatory responses to pathogens. Parameters determining the specificity and extent of mRNA degradation within the entire inflammation-associated transcriptome remain incompletely understood. Using transcriptome-wide high resolution occupancy assessment of the mRNA-destabilizing protein TTP, a major inflammation-limiting factor, we qualitatively and quantitatively characterize TTP binding positions and functionally relate them to TTP-dependent mRNA decay in immunostimulated macrophages. We identify pervasive TTP binding with incompletely penetrant linkage to mRNA destabilization. A necessary but not sufficient feature of TTP-mediated mRNA destabilization is binding to 3’ untranslated regions (UTRs). Mapping of binding positions of the mRNA-stabilizing protein HuR in activated macrophages revealed that TTP and HuR binding sites in 3’ UTRs occur mostly in different transcripts implicating only a limited co-regulation of inflammatory mRNAs by these proteins. Remarkably, we identify robust and widespread TTP binding to introns of stable transcripts. Nuclear TTP is associated with spliced-out introns and maintained in the nucleus throughout the inflammatory response. Our study establishes a functional annotation of binding positions dictating TTP-dependent mRNA decay in immunostimulated macrophages. The findings allow navigating the transcriptome-wide landscape of RNA elements controlling inflammation. PAR-iCLIP for TTP (3 replicates) and for HuR (2 replicates)
Project description:Individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) combined with high-throughput sequencing was performed to generate genome-wide binding maps of two U1-snRNP proteins: U1C and U1-70K in Trypanosoma brucei. 3 (2) biological replicates of U1C (U1-70K) -specific co-immunoprecipitated RNA after UV-crosslinking
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:APOBEC3G and APOBEC3F are cell-encoded restriction factors that have evolved to counteract virus infections. When packaged into HIV-1 particles, A3G and A3F impair reverse transcription and induce the hypermutation of viral DNA. We employed a next generation sequencing approach to identify the RNAs that these proteins bind in HIV-1 infected cells and HIV-1 virions. We then analysed the mechanism of packaging of APOBEC3 in detail.
Project description:A mutation within FASTKD2 causes a rare form of Mendelian mitochondrial encephalomyopathy. To investigate whether and how RNA binding of FASTKD2 contributes to the disease phenotype, we identified the RNA targets of FASTKD2 by iCLIP.
Project description:We mapped CstF64-RNA interactions at the transcriptome level and studied CstF64-mediated regulation of global mRNA alternative polyadenylation by using iCLIP-seq and direct RNA sequencing analyses. CstF64 iCLIP-seq in HeLa cells; direct RNA sequencing (DRS) of control HeLa cells, CstF64-RNAi cells and CstF64&CstF64tau-RNAi cells