Project description:Study of HP1 Knock Down on gene expression and splicing regulation in Human HeLa cells Maturation of pre-mRNAs is initiated co-transcriptionally. It is therefore conceivable that chromatin-borne information participates in alternative splicing. Here, we find that elevated levels of tri-methylation of histone H3 on lysine 9 (H3K9me3) are a characteristic of the alternative exons of several genes including CD44. On this gene, the chromodomain protein HP1gamma, frequently defined as a transcriptional repressor, facilitates inclusion of the alternative exons via a mechanism involving decreased RNA polymerase II elongation rate. In addition, accumulation of HP1gamma on the variant region of the CD44 gene stabilizes association of the pre-mRNA with the chromatin. Altogether, our data provide evidence for localized histone modifications impacting alternative splicing. They further implicate HP1gamma as a possible bridging molecule between the chromatin and the maturating mRNA, with a general impact on splicing decisions.
Project description:Analysis of the alternative pre-mRNA procesing after SF3b155 siRNA knock-down in HeLa cells employing a custom microarray platform sensitive to splicing. HeLa cells were transfected with either a scrambled RNA or one of the two different SF3b155 siRNA oligonucleotides (oligo 3: 5’-GACAGCAGAUUUGCUGGAUACGUGA-3; oligo 5: 5’-CCCUGUGGCAUUGCUUAAUGAUAU-3’). Total RNA samples from three biological replicates were labeled in direct and dye-swap hybridizations. Labeled samples were hybridized into our custom splicing-sensitive agilent platform which contains 1804 splicing events from 482 genes.
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of gene expression and alternative RNA splicing. The goals of this study are to compare alternative splicing in RALY knock-down cells to identify the function of RALY in alternative splicing transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis Methods: mRNA profiles of RALY knock-dowed HCT 116 cells and non-silencing shRNA treated HCT 116 cells generated by deep sequencing, in duplicate, using Illumina Hiseq 2500. Results: Using an we mapped about more than 60 million reads per sample to the human genome (GRCh38) and identified 58,884 transcripts in WT and RALY knock-downed HCT 116 cells with Hisat2. rMATS analysis of RNA-seq data demonstrate significant effects of RALY on 4,046 skipped exon splicing and other alternative splicing events. Conclusions: Our study represents the first detailed analysis of transcriptomes in RALY KD cells, with biologic duplicates, generated by RNA-seq technology. The splicing analysis workflows reported here should provide a framework for the RALY function in the splicing.
Project description:Study of HP1 Knock Down on gene expression and splicing regulation in Human HeLa cells Maturation of pre-mRNAs is initiated co-transcriptionally. It is therefore conceivable that chromatin-borne information participates in alternative splicing. Here, we find that elevated levels of tri-methylation of histone H3 on lysine 9 (H3K9me3) are a characteristic of the alternative exons of several genes including CD44. On this gene, the chromodomain protein HP1gamma, frequently defined as a transcriptional repressor, facilitates inclusion of the alternative exons via a mechanism involving decreased RNA polymerase II elongation rate. In addition, accumulation of HP1gamma on the variant region of the CD44 gene stabilizes association of the pre-mRNA with the chromatin. Altogether, our data provide evidence for localized histone modifications impacting alternative splicing. They further implicate HP1gamma as a possible bridging molecule between the chromatin and the maturating mRNA, with a general impact on splicing decisions. Transcriptome analysis of siRNA anti-HP1gamma transfected HeLa cells on GeneChip® Human Exon 1.0 ST Arrays (Affymetrix). Control HeLa cells have been transfected with the same concentration of siRNA anti-GAPDH. Experiment has been done in experimental triplicates.
Project description:We generated the SRSF2-/- Huh7 cells,and collected the cells at 2 days after transfected with siRNA. Then, we extracted RNAs and performed next generation sequencing. By comparing sequencing data from control and SRSF2 -/- samples, we profiled the alternative splicing events and gene expression regulated by SRSF2 in HCC.
Project description:We generated the liver specific SRSF2 -/- mice, and collected their liver at 11 day after birth and controls. Then, we extracted RNAs and performed next generation sequencing. By comparing sequencing data from WT and SRSF2 -/- samples, we profiled the alternative splicing events and gene expression regulated by SRSF2 during mouse liver development process.
Project description:Analysis of the alternative pre-mRNA procesing after SF3b155 siRNA knock-down in HeLa cells employing a custom microarray platform sensitive to splicing.
Project description:To determine targets of PTBP2-dependent alternative splicing, we depleted PTBP2 in human neurons derived from induced-pluripotent stem cells (iPSC-neurons) using an LNA gapmer and performed RNA-seq on untreated, negative control-treated, and knock-down samples.
Project description:We generated the liver specific SRSF2 -/- mice, and collected their liver at 5 month after born and controls. Then, we extracted RNAs and performed next generation sequencing. By comparing sequcing data from WT and SRSF2 -/- samples, we profiled the alternative splicing events and gene expression regulated by SRSF2 during mouse liver development process.