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RNA-Seq of EIF4A3 knockdown in human embryonic kidney cell line HEK293 via siRNA

ABSTRACT: RNA-Seq of EIF4A3 knockdown in human embryonic kidney cell line HEK293 via siRNA

PROVIDER: PRJEB72549 | ENA |

REPOSITORIES: ENA

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Dataset's files

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			Action	DRS
	ERR12652958_1.fastq.gz	Fastqsanger.gz
	ERR12652958_2.fastq.gz	Fastqsanger.gz
	ERR12652959_1.fastq.gz	Fastqsanger.gz
	ERR12652959_2.fastq.gz	Fastqsanger.gz
	ERR12652960_1.fastq.gz	Fastqsanger.gz

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U2OS LC MSMS upon siRNA-eIF4A3 or 5nM ActD treatment.

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Effects of nicastrin (NCSTN) siRNA knockdown in HEK293 and HEK001 cells.

Project description:The goal of this study was to determine if knockdown of nicastrin induced a proinflammatory phenotype in HEK001 and HEK293 cells. Nicastrin (NCSTN) is a member of the gamma-secretase complex, and has been identified as the most frequently mutated gene in familial hidradenitis suppurativa. While much research has been done into the effects of PSEN1 and PSEN2 loss, less is known about isolated NCSTN haploinsufficiency. Two cell lines were knocked down with either NCSTN siRNA or an siRNA to luciferase in triplicate. RNA was extracted from drug selected knockdowns and profiled on the Illumina Human HT-12 v4 beadarray. Gene ontology analysis of differentially expressed genes revealed a proinflammatory and decreased proliferation signature in keratinocytes. HEK293 cells demonstrated expression signatures for decreased cholesterol synthesis and interferon-alpha signaling, as well as increased p53 signaling and caspase mediated cytoskeletal cleavage. 12 total samples. Two cell lines (HEK001 & HEK293) each with two treatments (NCSTN siRNA knockdown or pLKO luciferase knockdown) in three parallel 3 replicates each. For each line gives the changes specific to knockdown of gamma-secretase component nicastrin (NCSTN).

2014-11-26 | E-GEOD-57949 | biostudies-arrayexpress

RNA-Seq of control and EJC factor (EIF4A3, RBM8A, MAGOH) knockdowns in part with EIF4A3 rescue in HeLa Tet-Off cells

Project description:The exon junction complex is involved in gene expression regulation on multiple co- and post-transcriptional levels. We aimed to investigate in HeLa Tet-Off cells which gene expression alterations can be observed upon knockdown of core EJC factors (EIF4A3, RBM8A and MAGOH) or upon EIF4A3 knockdown and rescue with EIF4A3 wild type construct by using RNA-Seq analyses. The rescue construct was stably integrated into the genome using the PiggyBac transposon system. As control Luciferase (Luc) siRNA was used.

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PRPF8 iCLIP on HeLa cells in response to eIF4A3 Knockdown

Project description:This experiment uses iCLIP to identify the binding pattern of the spliceosomal protein PRPF8 on RNA. The data shows that PRPF8 binds strongly and specifically in the region 12 to 14nt upstream of 5' splice sites (5ss). Due to PRPF8's role in the formation of the catalytically active spliceosome, this data can be used as a readout of 5ss selection. Here, we performed iCLIP on HeLa cells treated with control or EIF4A3 siRNA, with 4 replicate samples per condition and eIF4A3 protein levels reduced ~50% in knockdown. We investigated the role of the exon junction complex (EJC) in suppressing 5ss that are reconstituted at the junction of two canonical exons (RS-5ss) - selection of these splice sites would result in recursive splicing of canonical exons. We plotted the crosslink sites of reads that span an exon-exon junction, seperating reads that span RS-5ss from those that do not. We found that reads that span an RS-5ss are enriched at the 12-14nt window associated with 5ss selection, while reads that span other exon-exon junctions are not enriched. This effect is magnified greatly by knockdown of eIF4A3. The results indicate that RS-5ss can be used by the spliceosome, but that this process is usually repressed by the EJC. This data is evidence of recursive splicing of canonical exons and the role of the EJC in repressing recursive splicing.

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Project description:A loss of StarPap would be predicted to result in a decrease in cellular levels of mRNAs which it polyadenylates. Moreover, if PIPKIalpha has a function relationship with StarPap, knockdown of PIPKIalpha should cause a decrease in a pool of target mRNAs which require both StarPap and PIPKIalpha for their maturation. To test this, we independently knocked down StarPap and PIPKIalpha, and performed microarray analysis of total polyadenylated mRNAs from each group. Experiment Overall Design: HEK293 cells were transfected with siRNA specific for StarPap or PIPKIalpha or control siRNA. N=3

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Effects of nicastrin (NCSTN) siRNA knockdown in HEK293 and HEK001 cells.

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