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3096647.Pdf (1.038Mb) An shRNA-Based Screen of Splicing Regulators Identifies SFRS3 as a Negative Regulator of IL-1β Secretion The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Moura-Alves, Pedro, Ana Neves-Costa, Helena Raquel, Teresa Raquel Pacheco, Bruno D'Almeida, Raquel Rodrigues, Iris Cadima- Couto, et al. 2011. An shRNA-based screen of splicing regulators identifies SFRS3 as a negative regulator of IL-1β secretion. PLoS ONE 6(5): e19829. Published Version doi:10.1371/journal.pone.0019829 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:8296041 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA An shRNA-Based Screen of Splicing Regulators Identifies SFRS3 as a Negative Regulator of IL-1b Secretion Pedro Moura-Alves1., Ana Neves-Costa1., Helena Raquel1., Teresa Raquel Pacheco1, Bruno D’Almeida1, Raquel Rodrigues1, Iris Cadima-Couto1,Aˆ ngelo Chora1, Mariana Oliveira2, Margarida Gama-Carvalho2,3, Nir Hacohen4,5, Luis F. Moita1* 1 Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal, 2 Centro de Biodiversidade, Geno´mica Funcional e Integrativa (BioFIG), Faculdade de Cieˆncias, Universidade de Lisboa, Lisboa, Portugal, 3 Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal, 4 Division of Rheumatology, Allergy and Immunology, Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, United States of America, 5 Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America 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-1b secretion in response to E. coli challenge of THP-1 cells. Twelve of these genes are required for IL-1b secretion, while seven are negative regulators of this process. Silencing of SFRS3 increased IL-1b secretion due to elevation of IL-1b 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. Citation: Moura-Alves P, Neves-Costa A, Raquel H, Pacheco TR, D’Almeida B, et al. (2011) An shRNA-Based Screen of Splicing Regulators Identifies SFRS3 as a Negative Regulator of IL-1b Secretion. PLoS ONE 6(5): e19829. doi:10.1371/journal.pone.0019829 Editor: Juan Valcarcel, Centre de Regulacio´ Geno`mica, Spain Received October 27, 2010; Accepted April 18, 2011; Published May 17, 2011 Copyright: ß 2011 Alves et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: L.F.M. is a Young Investigator from the Human Frontier Science Program (www.hfsp.org) and receives support from Fundac¸a˜o Luso-Americana para o Desenvolvimento (http://www.flad.pt/) and Fundac¸a˜o para a Cieˆncia e a Tecnologia (http://alfa.fct.mctes.pt/); (PTDC/SAU-MII/100780/2008 and PIC/IC/82991/ 2007). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] . These authors contributed equally to this work. Introduction functionally significant in the immune and nervous systems [4,10]. The success of the vertebrate immune system relies on a Microorganisms activate the innate immune system using remarkable potential to generate highly diverse detection, germline-encoded pattern-recognition receptors (PRRs). Several transduction and effector mechanisms in addition to the ability classes of PRRs, including Toll-like receptors, recognize distinct of individual cells to rapidly adapt and respond to changing microbial components and directly activate immune cells, environmental conditions [1]. Transcriptional regulation in the including antigen presentation cells such as dendritic cells (DCs). immune system has received the most attention in recent years, Exposure of immune cells to the agonists of these receptors but achieving such diversity and flexibility of function requires the activates the NF-kB pathway, rapidly inducing the expression of operation of additional mechanisms of gene regulation. Alternative cytokines (such as IL-1b), co-stimulatory molecules and other Splicing (AS), which affects most of human genes and is altered in effector molecules that are part of an effective immune response at least 15% of all point mutations causing human genetic disease [1]. This initial response is terminated several hours later through [2,3], is a potential critical mechanism for the regulation and fine- incompletely characterized molecules and pathways [11] but that tuning of physiological immune responses [4,5]. are of critical importance to avoid an excessive NF-kB signaling. Our genome contains much fewer coding genes than anti- The NF-kB transcription factor has been the subject of intense cipated before the completion of the human genome-sequencing study for many years, reflecting its importance in the immune project [6]. Alternative splicing permits the generation of a large response. Interestingly, many components along the pathways array of mRNA transcripts and protein isoforms from a limited leading to NF-kB activation, such as MyD88, IRAK family number of genes [4,5]. By including or deleting functional protein members, TAB1, TAK1, IkB kinase complex factors and NF-kB domains it can change many protein properties including transcription factors (reviewed in [5]) have been shown to undergo protein–protein interactions, subcellular localization, stability, induced alternative splicing in the later phase of the immune DNA binding, and enzymatic properties of the dominant response and to generate shorter isoforms that can act as dominant isoforms [7]. Two recent landmark reports estimate that 92% negative factors able to shut down NF-kB dependent transcription. to 95% of all human primary transcripts can undergo alternative Here we report the use of a subset of the TRC lentiviral human splicing [8,9]. This process seems to be especially prevalent and library [12] to generate loss-of-function phenotypes for a large PLoS ONE | www.plosone.org 1 May 2011 | Volume 6 | Issue 5 | e19829 SFRS3 Is a Negative Regulator of IL-1b Secretion collection of genes known or predicted to be involved in the (qRT-PCR), total RNA was extracted using TRIzol reagent splicing reaction (425 genes), with an average 5-fold coverage [13]. (Invitrogen) and cDNA synthesis used Superscript II Reverse By applying this library in human THP-1 monocytic cells, we aim Transcriptase (Invitrogen). qRT-PCR was performed in the to systematically identify components of the splicing machinery presence of Power SYBR green PCR Master Mix (Applied that modulate the secretion of IL-1b. We have identified 19 genes Biosystems) and the amplification protocol was performed that significantly affect the production of IL-1b after a 24 h on a Rotor-Gene 6000 (Corbett). All samples were normalized challenge with PFA-fixed E. coli. Twelve of these genes are to the expression of glyceraldehyde-3-phosphate dehydrogenase required for IL-1b secretion, while seven are negative regulators of (GAPDH) and relative expression was calculated using Pfaffl’s this process. We chose one of the highest scoring negative method [17]. regulators, SFRS3, for further characterization of its role in IL-1b For immunoblotting (IB), cells were collected, washed twice and secretion. lysed for 15 minutes at 4uC using RIPA buffer (50 mM Tris-HCl at pH = 7.4, 1% NP-40, 0.25% Sodium Deoxicholate, 150 mM Methods NaCl, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF in the presence of proteases inhibitor cocktail (Roche)). Primary antibodies against Cell Culture b-actin, caspase-1, HMGB1, NLRP3 and V5 were from Abcam; THP-1 cells (ATCC TIB-202) were grown in R10- RPMI against ASC and SFRS3 were from Abnova. Secondary antibodies media 1640 supplemented with 10% (v/v) Fetal Bovine Serum, against mouse or rabbit were from Cell Signaling. Caspase-1 1% (v/v) Penicillin-Streptomycin, 1% (v/v) Pyruvate , 1% (v/v) L- activity was measured using the Carboxyfluorescein FLICA Glutamine , 1% (v/v) Non-essential aminoacids , 1% (v/v) Hepes Detection kit for Caspase Assay (Immunochemistry Technologies, buffer and 0.05 M of 2-Mercaptoethanol, HEK 293T cells LLC). Briefly, cells were incubated for 1 hour at 37uC with 306 (ATCC CRL-11268) were grown in Dulbecco’s modified Eagle’s FLICA solution at a 1:30 ratio, washed 3 times, and ressuspended medium (DMEM) supplemented with 10% (v/v) Fetal Bovine in 150 mL of wash buffer. The samples were immediately assayed Serum, 1% (v/v) Penicillin-Streptomycin and 0.05 M of 2- by Flow citometry, using a FACSCalibur system (BD biosciences) Mercaptoethanol. Cells were kept at 37uC under a 5% carbon and the data was analysed using FlowJo software (Tree Star Inc). dioxide (CO2) atmosphere. Purification of bone marrow derived cells (BMDCs) was adapted from previously described [14,15]. SFRS3 Overexpression Briefly, marrow cavities of the tibias and femurs of 8–12 week-old mice were flushed with complete RPMI 1640 (10% FBS) using a Lentiviral vector for SFRS3 protein overexpression was 27-gauge needle (Terumo, Tokyo, Japan). After red cells constructed using the Gateway pENTR11 and pLenti6.2/V5- hypotonic lysis for 5 min (with ammonium chloride solution), DEST Gateway vectors from Invitrogen.
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