ICLIP mapping of coilin-RNA interactions in HeLa and P19 cells
ABSTRACT: 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:We used GFP-tagged SR proteins expressed at endogenous levels and iCLIP to identify and compare endogenous RNA targets of individual SR proteins, map the preferential sites of binding, compare binding pattern and binding motifs between family members and to NXF1 and quantify binding of SR proteins and NXF1 to spliced versus unspliced RNAs to study the role of SR proteins in mRNA export via NXF1. Overall design: For each GFP-tagged SR proteins and NXF1, 3 to 6 biological replicates were subjected to the iCLIP procedure with UV-crosslinking at 254 nm. An anti-GFP antibody as used for IPs. As negative control served samples prepared from a P19 cell line expressing only GFP-NLS fusion protein.
Project description:SIRT7 is an NAD+-dependent protein deacetylase with important roles in ribosome biogenesis and cell proliferation. Previous studies have established that SIRT7 is associated with RNA polymerase I, interacts with pre-rRNA and promotes rRNA synthesis. Here we show that SIRT7 is also associated with snoRNAs that are involved in pre-rRNA processing and rRNA maturation. Knockdown of SIRT7 impairs U3 snoRNA-dependent early cleavage steps that are necessary for generation of 18S rRNA. Mechanistically, SIRT7 deacetylates U3-55k, a core component of the U3 snoRNP complex, and reversible acetylation of U3-55k modulates the association of U3-55k with U3 snoRNA. Deacetylation by SIRT7 enhances U3-55k binding to U3 snoRNA, which is a prerequisite for pre-rRNA processing. Under stress conditions, SIRT7 is released from nucleoli, leading to hyperacetylation of U3-55k and attenuation of prerRNA processing. The results reveal a multifaceted role of SIRT7 in ribosome biogenesis, regulating both transcription and processing of rRNA. CLIP-seq was performed in Flag-SIRT7-293T cells.
Project description:HeLa cells were cultured in DMEM, supplemented with 10% (v/v) FCS and penicillin/streptomycin under 5% CO2 at 37°C. For iCLIP, HeLa cells expressing eIF4A3-GFP or PTB-GFP were induced with doxycycline to adjust the level of recombinant protein to the level of the endogenous counterpart and irradiated with 150 mJ/cm2 UV light (254 nm). The iCLIP cDNA libraries for eIF4A3 and PTB were sequenced with 50 bp on an Illumina HiSeq instrument.
Project description:MicroRNA miRNA expression profiles for human HeLa cells (Cervical cancer), overexpressing p19 H-Ras, were examined to investigate the miRNA regulation by p19 H-Ras. miRNA microarray analysis identified statistical unique profiles, which could discriminate miRNAs regulated by p19 H-Ras and not regulated by the p19 mutant (W164A) H-Ras.
Project description:HeLa cells were cultured in DMEM, supplemented with 10% (v/v) FCS and penicillin/streptomycin under 5% CO2 at 37C. For iCLIP, HeLa cells expressing GFP fusion proteins were induced with doxycycline to adjust the level of recombinant protein to the level of the endogenous counterpart and irradiated with 150 mJ/cm2 UV light (254 nm). The iCLIP cDNA libraries were sequenced with 50 bp on an Illumina HiSeq 2000 instrument. RNASeq was performed as a control with 50 bp paired-end on an Illumina HiSeq 2000 instrument.
Project description:We performed iCLIP of CPSF-160- GFP as CPSF160 was believed to be the main RNA-binding subunit of CPSF. To this end, we used a stable HeLa cell line that expresses GFP-CPSF160 in a doxycycline (Dox)-dependent manner. After GFP-CPSF160 was induced, we irradiated the cells with UV-C (wavelength=254nm) to induce protein-RNA crosslinks and immunoprecipitated the complex with a GFP antibody.
Project description:To investigate whether Rbfox3 could alter the expression level of miRNAs during neuronal differentiation of P19 cells, we performed miRNA microarray analysis using the RNAs extracted from untreated (undifferentiated) P19-GFP, RA-treated (neuronally differentiated) P19-GFP, or RA-treated P19-T2 cells. Total 9 samples were analyzed. We compared expression levels of P19-GFP (-) vs P19-GFP (+) vs P19-T2 (+) to identify miRNAs which had changes in expression levels with p < 0.01. From this miRNA list, we compared among P19-GFP (-) vs P19-GFP (+) vs P19-T2 (+) to identify the miRNAs which appeared to correlate with Rbfox3 expression.
Project description:In eukaryotes, biogenesis of ribosomes requires folding and assembly of the precursor rRNA (pre-rRNA) with a large number of proteins and snoRNPs into huge RNA-protein complexes. In spite of intense genetic, biochemical and high resolution cryo-EM studies in Saccharomyces cerevisiae, information about the conformation of the earliest 35S pre-rRNA is limited. To overcome this, we performed high-throughput SHAPE chemical probing on the 35S pre-rRNA associated with 90S pre-ribosomes. We focused our analyses on external (5´ETS) and internal (ITS1) transcribed spacers as well as the 18S region. We show that in the 35S pre-rRNA, the central region of the 18S is in a more open configuration compared to 20S pre-rRNA and that the central pseudoknot is not formed. The essential ribosome biogenesis protein Mrd1 influences the structure of the 18S part locally and is involved in organizing the central pseudoknot and surrounding structures. Our results demonstrate that the U3 snoRNA dynamically interacts with the 35S pre-rRNA and that Mrd1 is required for disrupting U3 snoRNA base-pairing interactions in the 5'ETS. We propose that the dynamic U3 snoRNA interactions and Mrd1 are essential for establishing the structure of the central region of 18S that is required for processing and 40S subunit function. Overall design: Two different ribosomal RNA species were probed with 1M7 This series contains 4 samples re-analyzed from GSE83821. The raw data associated with these samples in this series (GSM2856218-GSM286221) include the barcodes not present in the raw data from the original samples from GSE83821 (GSM2219115-GSM2219118).