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An shRNA-based screen of splicing regulators identifies SFRS3 as a negative regulator of IL-1? secretion.

ABSTRACT: The generation of diversity and plasticity of transcriptional programs are key components of effective vertebrate immune responses. The role of Alternative Splicing has been recognized, but it is underappreciated and poorly understood as a critical mechanism for the regulation and fine-tuning of physiological immune responses. Here we report the generation of loss-of-function phenotypes for a large collection of genes known or predicted to be involved in the splicing reaction and the identification of 19 novel regulators of IL-1? secretion in response to E. coli challenge of THP-1 cells. Twelve of these genes are required for IL-1? secretion, while seven are negative regulators of this process. Silencing of SFRS3 increased IL-1? secretion due to elevation of IL-1? and caspase-1 mRNA in addition to active caspase-1 levels. This study points to the relevance of splicing in the regulation of auto-inflammatory diseases.

SUBMITTER: Moura-Alves P

PROVIDER: S-EPMC3096647 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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An shRNA-based screen of splicing regulators identifies SFRS3 as a negative regulator of IL-1β secretion.

Moura-Alves Pedro P Neves-Costa Ana A Raquel Helena H Pacheco Teresa Raquel TR D'Almeida Bruno B Rodrigues Raquel R Cadima-Couto Iris I Chora Ângelo Â Oliveira Mariana M Gama-Carvalho Margarida M Hacohen Nir N Moita Luis F LF

PloS one 20110517 5

The generation of diversity and plasticity of transcriptional programs are key components of effective vertebrate immune responses. The role of Alternative Splicing has been recognized, but it is underappreciated and poorly understood as a critical mechanism for the regulation and fine-tuning of physiological immune responses. Here we report the generation of loss-of-function phenotypes for a large collection of genes known or predicted to be involved in the splicing reaction and the identificat ...[more]

PMID: 21611201

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Project description:BackgroundThe molecular regulators that orchestrate stem cell renewal, proliferation and differentiation along the mammary epithelial hierarchy remain poorly understood. Here we have performed a large-scale pooled RNAi screen in primary mouse mammary stem cell (MaSC)-enriched basal cells using 1295 shRNAs against genes principally involved in transcriptional regulation.MethodsMaSC-enriched basal cells transduced with lentivirus pools carrying shRNAs were maintained as non-adherent mammospheres, a system known to support stem and progenitor cells. Integrated shRNAs that altered culture kinetics were identified by next generation sequencing as relative frequency changes over time. RNA-seq-based expression profiling coupled with in vitro progenitor and in vivo transplantation assays was used to confirm a role for candidate genes in mammary stem and/or progenitor cells.ResultsUtilizing a mammosphere-based assay, the screen identified several candidate regulators. Although some genes had been previously implicated in mammary gland development, the vast majority of genes uncovered have no known function within the mammary gland. RNA-seq analysis of freshly purified primary mammary epithelial populations and short-term cultured mammospheres was used to confirm the expression of candidate regulators. Two genes, Asap1 and Prox1, respectively implicated in breast cancer metastasis and progenitor cell function in other systems, were selected for further analysis as their roles in the normal mammary gland were unknown. Both Prox1 and Asap1 were shown to act as negative regulators of progenitor activity in vitro, and Asap1 knock-down led to a marked increase in repopulating activity in vivo, implying a role in stem cell activity.ConclusionsThis study has revealed a number of novel genes that influence the activity or survival of mammary stem and/or progenitor cells. Amongst these, we demonstrate that Prox1 and Asap1 behave as negative regulators of mammary stem/progenitor function. Both of these genes have also been implicated in oncogenesis. Our findings provide proof of principle for the use of short-term cultured primary MaSC/basal cells in functional RNAi screens.

Project description:ObjectivesGlucose-stimulated insulin secretion is a critical function in the regulation of glucose homeostasis, and its deregulation is associated with the development of type 2 diabetes. Here, we performed a genetic screen using islets isolated from the BXD panel of advanced recombinant inbred (RI) lines of mice to search for novel regulators of insulin production and secretion.MethodsPancreatic islets were isolated from 36 RI BXD lines and insulin secretion was measured following exposure to 2.8 or 16.7 mM glucose with or without exendin-4. Islets from the same RI lines were used for RNA extraction and transcript profiling. Quantitative trait loci (QTL) mapping was performed for each secretion condition and combined with transcriptome data to prioritize candidate regulatory genes within the identified QTL regions. Functional studies were performed by mRNA silencing or overexpression in MIN6B1 cells and by studying mice and islets with beta-cell-specific gene inactivation.ResultsInsulin secretion under the 16.7 mM glucose plus exendin-4 condition was mapped significantly to a chromosome 2 QTL. Within this QTL, RNA-Seq data prioritized Crat (carnitine O-acetyl transferase) as a strong candidate regulator of the insulin secretion trait. Silencing Crat expression in MIN6B1 cells reduced insulin content and insulin secretion by ?30%. Conversely, Crat overexpression enhanced insulin content and secretion by ?30%. When islets from mice with beta-cell-specific Crat inactivation were exposed to high glucose, they displayed a 30% reduction of insulin content as compared to control islets. We further showed that decreased Crat expression in both MIN6B1 cells and pancreatic islets reduced the oxygen consumption rate in a glucose concentration-dependent manner.ConclusionsWe identified Crat as a regulator of insulin secretion whose action is mediated by an effect on total cellular insulin content; this effect also depends on the genetic background of the RI mouse lines. These data also show that in the presence of the stimulatory conditions used the insulin secretion rate is directly related to the insulin content.

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An shRNA-based screen of splicing regulators identifies SFRS3 as a negative regulator of IL-1? secretion.

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An shRNA-based screen of splicing regulators identifies SFRS3 as a negative regulator of IL-1β secretion.

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