Hsa-miR-139-5p/HNRNPF axis modulates gene-transcripts balance in thyroid cancer cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE123680 | GEO | 2019/08/26
REPOSITORIES: GEO
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Project description:We reported the joint SRRM1/2 regulation is not the major mechanism of SRRM2-mediated alternative splicing, and that maintenance of splicing condensates by SRRM2 is important for proper alternative splicing. We knocked SRRM1/2 down with three different shRNAs, performed RNA-seq and derived the differential alternative splicing events (DASEs) using rMATS. DASEs of SRRM1/2 were largely different with little overlap, and displayed a different splicing pattern. SRRM2 deficiency induces skipping of cassette exons with short introns and weak 5’ splice. Nearly 25% of the skipped events upon SRRM2 knockdown were unannotated. More than 40% of validated DASEs upon SRRM2 suppression were associated with protein domain change (>50% domain removed by splicing). Domain changes, and especially large region removal by multiple exon skipping is a significant mechanism for protein dysfunction. Thus, alternative splicing is the major mechanism for the maintenance of protein function and cell homeostasis by SRRM2.
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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.
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Project description:Deletion of Dars2 in HSPCs largely impairs spliceosome function and suppresses the protein expression of Srsf3.We performed high-coverage mRNA-seq on lin- ckit+ (LK) cells from both WT and Dars2-KO mice, respectively, to characterize genome-wide alternative splicing (AS) events affected in Dars2-KO HSPCs. Globally, 3970 AS events were significantly changed by loss of Dars2 in HSPCs, including 2431 events in skipped exons (SEs), 516 events in retained introns (RIs), 375 events in mutually exclusive exons (MXEs), 340 events in alternative 3’ splice sites (A3SSs) and 308 events in alternative 5’ splice sites (A5SSs)
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Project description:It is generally thought that splicing factors regulate alternative splicing through binding to RNA consensus sequences. In addition to these linear motifs, RNA secondary structure is emerging as an important layer in splicing regulation. Here we demonstrate that RNA elements with G-quadruplex forming capacity promote exon inclusion. Destroying G-quadruplex forming capacity while keeping G-tracts intact abrogates exon inclusion. Analysis of RNA binding protein footprints revealed that G-quadruplexes are enriched in hnRNPF-binding sites and near hnRNPF-regulated alternatively spliced exons in the human transcriptome. Moreover, hnRNPF regulates an EMT-associated CD44 isoform switch in a G-quadruplex-dependent manner, which results in inhibition of EMT. Mining breast cancer TCGA datasets, we demonstrate that hnRNPF negatively correlates with an EMT gene signature and positively correlates with patient survival. These data suggest a critical role for RNA G-quadruplexes in regulating alternative splicing. Modulation of G-quadruplex structural integrity may control cellular processes important for tumor progression.
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Project description:E10.5 whole brain were isolated from WT and Piezo1-KO mice (C57BL/6J strain backgound) described in Ranade et al 2014. https://doi.org/10.1073/pnas.1409233111
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Project description:Purpose: this study is to analyze the change of RNA splicing events after disruption of Protein Arginine Methyltranferase 5 (PRMT5) in Plasmodium falciparum. Methods: In this study, the transcriptomes of a PfPRMT5 gene knockout (KO) parasite line with its wildtype control were analyzed by RNAseq.Total RNA were harvested from the asexual parasites at four stages (ring, early trophozoite, late trophozoite, and schizont)(12h, 24h, 36h, and 46h post-invasion) using the Quick-RNA MiniPrep kit (Zymo Research). RNA sequencing libraries were prepared using the KAPA stranded RNA-seq library preparation kit (Roche) with 500 ng RNA from each sample. Illumina adapter sequence removal and quality trimming of reads were performed using Trimmomatic. Only reads that had a minimum length of 50 base pairs were retained. Reads were then mapped to the P. falciparum 3D7 strain reference genome with HISAT2. Results: This RNAseq analysis with strand-specific mRNA libraries from both ΔPfPRMT5 and WT lines showed that >90% of sequencing reads were of high quality for mapping to the P. falciparum genome with nearly 40 times of coverage. This allows us to analyze the change of alternative splicing events (alternative 5' splice site, alternative 3' splice site, retained intron and skipped exon). 800, 1056, 479, and 1158 alternative splicing events (alternative 5' splice site, alternative 3' splice site, retained intron and skipped exon) were altered in the DPfPRMT5 parasite line as compared to the WT line at four development stages, respectively (unpublished data). Conclusions: Collectively, this RNAseq provide a dataset for analysis of abnormal RNA splicing events in the ΔPfPRMT5 parasites.
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Project description:Alternative splicing generates functional diversity in higher organisms through alternative first and last exons, skipped and included exons, intron retentions and alternative donor and acceptor sites. In large-scale microarray studies in human and mouse, emphasis so far has been placed on exon-skip events, leaving the prevalence and importance of other splice types largely unexplored. Using a new human splice variant database and a genome-wide microarray to probes thousands of splice events of each type, we measured differential expression of splice types across 6 pairs of diverse cell lines and validated the database annotation process. Results suggest that splicing in human is more complex than simple exon skip events, which account for a minority of splicing differences. The relative frequency of differential expression of the splice types correlates with what is found by our annotation efforts. In conclusion, alternative splicing in human cells is considerably more complex than the canonical example of the exon-skip. The complementary approaches of genome-wide annotation of alternative splicing in human and design of genome-wide splicing microarrays to measure differential splicing in biological samples provide a powerful high-throughput tool to study the role of alternative splicing in human biology. Keywords: alternative splicing
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Project description:Alternative splicing of RNA transcripts can result in a multitude of variations from the genome. Since splicing can occur co-transcriptionally, chromatin structure has been reported to affect the choice of a splice site. TIP60 is a haploinsufficient tumor suppressor gene that is frequently downregulated in cancers. Since TIP60 is a writer of histone and non-histone modifications, we investigated TIP60’s role in modulating alternative splicing. RNA sequencing was performed in TIP60-depleted MCF10A cells and with cells rescued with wild-type or catalytically inactive TIP60. The majority of the differential splicing events were skipped exon events, including exon 25 skipping event of ITGA6. To identify the splicing factor that regulates this event, a siRNA screen of 77 splicing factors was employed. TIP60 depletion results in an increase in the ITGA6 isoform that lacks exon 25 and the siRNA screen identified ESRP2 to regulate splicing of ITGA6 exon 25. This study suggests that alternative splicing of ITGA6 exon 25 is regulated by the TIP60-ESRP2 axis.
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Project description:Alternative splicing (AS) plays an important role in the development and activation of B cells. RNA-binding proteins (RBPs) have emerged as critical players in AS regulation. RNA-binding protein hnRNPF regulates AS of several important genes, such as c-Src, Bcl-xl, Mcl-1 and Tcf3 in tumor cells, but its function in B cell biology is not known.Here we constructed CD19-Cre mediated hnRNPF conditional knockou mice and performed RNA-sequencing to profile mRNA expression and AS regulated by hnRNPF in splenic B cells.
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Project description:Purpose: The goal of this study was to determine squalene effect on hepatic transcriptome by comparing high-throughput data for tested group vs. control group in male Large White x Landrace Large swine animals Method: Study the Hepatic mRNA profile of swine animals in response to a steatotic diet supplied with 0.5% squalene over a month by Transcriptome sequencing using DNBseq platform. Sequence reads that passed quality filters were mapped onto reference genome, followed by SNP & INDEL variants calling and Gene expression detection. Results: Squalene intake did not significantly influence single nucleotide polymorphisms. When alternative splicing events were tested, squalene administration had significant influence on splicing events including alternative 5' splicing, alternative 3' splicing , retained intron, skipped exons and mutually exclusive exons. Splicing patterns led to variety of differentially splicing genes and a variety of different isoforms from one gene. Differentially expressed genes were 18,659 in the squalene group and 18,602 in control group.
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