Identification of SRSF9 through pooled shRNA screening links BNIP3 splicing to autophagy and metabolic reprogramming in breast cancer
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PROVIDER: PRJNA1248513 | ENA |
REPOSITORIES: ENA
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Project description:Autophagy is a catabolic process that facilitates the degradation of toxic molecules and contributes to the maintenance of cellular homeostasis. However, the alternative splicing of genes involved in hypoxia-induced autophagy remains relatively unexplored. Here we performed pooled shRNA screening of multiple RNA binding proteins under hypoxia to mechanistically decipher their role in regulating alternative splicing of autophagy genes in breast cancer cells. We reported reduced SRSF9 expression under hypoxia that when exogenously expressed diminishes autophagosome formation. SRSF9 regulates alternative splicing of BNIP3 generating two functionally distinct isoforms regulating autophagosome formation and cancer progression through Warburg effect. Mechanistically, SRSF9 directly binds to exon 3 of BNIP3 mRNA inhibiting the truncated BNIP3- Δ3 generation and resultant warburg effect. Moreover, the individual ectopic expression of full length BNIP-FL promotes autophagosome formation. Collectively, our study identifies novel mechanism where SRSF9 mediates the switch between autophagy and Warburg effect via BNIP3 alternative splicing thereby impacting breast cancer progression and chemoresistance.
2025-07-11 | GSE294174 | GEO
Project description:shRNA knockdown against SRSF9 in HepG2 cells followed by RNA-seq. (SRSF9-BGHLV12) For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODE_Data_Use_Policy_for_External_Users_03-07-14.pdf
2016-05-26 | GSE80907 | GEO
Project description:shRNA knockdown against SRSF9 in K562 cells followed by RNA-seq. (SRSF9-LV11) For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODE_Data_Use_Policy_for_External_Users_03-07-14.pdf
2016-05-26 | GSE80856 | GEO
Project description:Here, we show that the splicing factor SRSF9 selectively controls the editing of many brain-specific sites in mammals. SRSF9 is more lowly expressed in the mammalian brain than in non-brain tissues. Gene perturbation experiments and minigene analysis of candidate sites demonstrated that SRSF9 could robustly repress A-to-I editing by ADAR2. We found that SRSF9 biochemically interacted with ADAR2 in the nucleus via its RRM2 domain alone. Additionally, this interaction required the presence of the RNA substrate and disrupted the formation of ADAR2 dimers. Using transcriptome-wide location analysis and genome-wide RNA profiling, we identified 1,328 editing sites that are regulated directly by SRSF9. Importantly, we showed that this SRSF9 regulon is significantly enriched for brain-specific editing sites despite our unbiased approach. We further provide evidence that SRSF9 prevents cells from undergoing apoptosis possibly through the inhibition of ADAR2-mediated editing. Collectively, our results highlighted the importance of SRSF9 as an editing regulator and suggested that other splicing factors may also be involved in regulating RNA editing.
2018-06-21 | GSE108125 | GEO
Project description:The goal of the study was to characterize the mechanism by which Raptinal induces apoptosis using a genome-wide pooled shRNA screen. Following drug treatment, the abundance of the shRNA constructs were determined and compared between DMSO and Raptinal-treated samples. Constructs depleted in Raptinal samples versus DMSO control, are expected to potentiate Raptinal-induced apoptosis by down regulating anti-apoptotic proteins. In contrast, shRNA constructs enriched upon Raptinal treatment are expected to target pro-apoptotic proteins, whose down regulation confers protection from Raptinal-induced apoptosis. Follow up experiments were performed on enriched shRNAs in order to validate potentially novel pro-apoptotic proteins.
2018-02-27 | GSE66423 | GEO
Project description:Adenosine DeAminases acting on RNA (ADAR) catalyzes adenosine-to-inosine (A-to-I) conversion within RNA duplex structures. While A-to-I editing is often dynamically regulated in a spatial-temporal manner, the mechanisms underlying its tissue-selective restriction remain elusive. We have previously reported that transcripts of voltage-gated calcium channel CaV1.3 are subject to brain-selective A-to-I RNA editing by ADAR2. Here, we show that editing of CaV1.3 mRNA is dependent on a 40 bp RNA duplex formed between exon 41 and an evolutionarily conserved editing site complementary sequence (ECS) located within the preceding intron. Heterologous expression of a mouse minigene that contained the ECS, intermediate intronic sequence and exon 41 with ADAR2 yielded robust editing. Interestingly, editing of CaV1.3 was potently inhibited by serine/arginine-rich splicing factor 9 (SRSF9). Mechanistically, the inhibitory effect of SRSF9 required direct RNA interaction. Selective down-regulation of SRSF9 in neurons provides a basis for the neuron-specific editing of CaV1.3 transcripts.
2018-04-17 | GSE113193 | GEO
Project description:Label the cells overexpressed Flag tagged YTHDC1, SRSF1, SRSF3, SRSF7, SRSF9 and SRSF10 with 4-SU, the RNA bound by YTHDC1 and SRSF proteins can be got by Flag IP followed by RNA isolation by using the TRIzol (Invitrogen) reagent by following the company manual.the RNA was then used for library preparation using a TruSeq⢠RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) according to the manufacturerâs protocol.The libraries were sequenced using HiSeq2000 (Illumina) in single-read mode, creating reads with a length of 101 bp. Sequencing chemistry v2 (Illumina) was used and samples were multiplexed in two samples per lane. Discovery of the binding motif of YTHDC1, SRSF1, SRSF3, SRSF7, SRSF9 or SRSF10 in overexpressed Human HeLa cells
2016-02-17 | E-GEOD-71096 | biostudies-arrayexpress
Project description:Purpose: A-to-I RNA editing is critical for many cellular processes. The sites of A-I RNA editing can be identified through RNA-seq and matching the reads to the annotated database like RADAR. The goals of this study is to compare A-I RNA editing profile among wild type, ADAR2 overexpressing and ADAR2+SRSF9 overexpressing 293T cells to evaluate the influence of SRSF9 repressive action on A-I RNA editing Methods: A-I RNA editing profile and gene expression profiles were generated by deep sequencing from all the samples, using NEBNext® Ultra™ Directional RNA Library Prep Kit. The sequence reads that passed quality filters were analyzed for identifying the A-I RNA editing sites using RADAR Database Results: Upon overexpression of ADAR2, we found that 18,174 sites were differentially edited compared to control cells, of which 97.0% showed a significant increase in their editing levels as expected (P < 0.05, Fisher’s test). Furthermore, when we overexpressed both ADAR2 and SRSF9 together, we found that 6,994 sites were differentially edited compared to overexpression of the deaminase alone (P < 0.05, Fisher’s test). Importantly, the editing of 92.5% of these sites was down-regulated, consistent with the function of SRSF9 as a repressor of editing. Conclusions: Our study reports a detailed analysis of the effect of SRSF9 on A-I RNA editing of ADAR2-specific targets. We Conclude that this SRSF9 regulon is significantly enriched for brain-specific editing sites despite our unbiased approach.
2018-06-21 | GSE114970 | GEO
Project description:Purpose: A-to-I RNA editing is critical for many cellular processes. The sites of A-I RNA editing can be identified through RNA-seq and matching the reads to the annotated database like RADAR. The goals of this study is to compare A-I RNA editing profile among wild type, ADAR2 overexpressing and ADAR2+SRSF9 overexpressing 293T cells to evaluate the influence of SRSF9 repressive action on A-I RNA editing Methods: A-I RNA editing profile and gene expression profiles were generated by deep sequencing from all the samples, using NEBNext® Ultra™ Directional RNA Library Prep Kit. The sequence reads that passed quality filters were analyzed for identifying the A-I RNA editing sites using RADAR Database Results: Upon overexpression of ADAR2, we found that 18,174 sites were differentially edited compared to control cells, of which 97.0% showed a significant increase in their editing levels as expected (P < 0.05, Fisher’s test). Furthermore, when we overexpressed both ADAR2 and SRSF9 together, we found that 6,994 sites were differentially edited compared to overexpression of the deaminase alone (P < 0.05, Fisher’s test). Importantly, the editing of 92.5% of these sites was down-regulated, consistent with the function of SRSF9 as a repressor of editing. Conclusions: Our study reports a detailed analysis of the effect of SRSF9 on A-I RNA editing of ADAR2-specific targets. We Conclude that this SRSF9 regulon is significantly enriched for brain-specific editing sites despite our unbiased approach.
2018-06-21 | GSE108126 | GEO
Project description:Memory B cells are long-lived lymphocytes essential for humoral immunological memory. Autophagy is required for the long-term survival of memory B cells, however, the molecular mechanisms for the protection by autophagy have not been fully elucidated. Here we show that mitochondrial autophagy mediated by Nix and Bnip3 prevents necroptosis in memory B cells by maintaining mitochondrial homeostasis and normal lipid metabolism. In mice with B cell-specific deletion of Nix and Bnip3, memory B cells showed significant increases in viable mitochondria and de novo fatty acid synthesis, with accumulation of lipid droplets in the cytoplasm and accelerated cell death. Inhibiting the mitochondrial citrate transporter Slc25a1 or silencing the necroptosis gene Ripk3 rescued Nix–/–Bnip3–/– memory B cells in vivo. These data indicate that Nix- and Bnip3-dependent mitochondrial autophagy in memory B cells is important for restricting de novo fatty acid synthesis and preventing necroptosis. Our results suggest a mechanism for autophagy in the maintenance of mitochondrial homeostasis for the protection of immunological memory.
2021-06-03 | GSE176047 | GEO