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Deletion of AU-Rich Elements Within the Bcl2 3′UTR Reduces Protein Expression and B Cell Survival in Vivo

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Deletion of AU-Rich Elements Within the Bcl2 3′UTR Reduces Protein Expression and B Cell Survival in Vivo RESEARCH ARTICLE Deletion of AU-Rich Elements within the Bcl2 30UTR Reduces Protein Expression and B Cell Survival In Vivo Manuel D. Díaz-Muñoz, Sarah E. Bell, Martin Turner* Lymphocyte Signalling and Development, The Babraham Institute, Cambridge, United Kingdom * [email protected] Abstract a11111 Post-transcriptional mRNA regulation by RNA binding proteins (RBPs) associated with AU- rich elements (AREs) present in the 30 untranslated region (3’UTR) of specific mRNAs mod- ulates transcript stability and translation in eukaryotic cells. Here we have functionally characterised the importance of the AREs present within the Bcl2 3’UTR in order to maintain Bcl2 expression. Gene targeting deletion of 300 nucleotides of the Bcl2 3’UTR rich in AREs diminishes Bcl2 mRNA stability and protein levels in primary B cells, decreasing cell life- OPEN ACCESS Δ Δ span. Generation of chimeric mice indicates that Bcl2-ARE / B cells have an intrinsic com- Citation: Díaz-Muñoz MD, Bell SE, Turner M (2015) petitive disadvantage compared to wild type cells. Biochemical assays and predictions Deletion of AU-Rich Elements within the Bcl2 30UTR Reduces Protein Expression and B Cell Survival In using a bioinformatics approach show that several RBPs bind to the Bcl2 AREs, including Vivo. PLoS ONE 10(2): e0116899. doi:10.1371/ AUF1 and HuR proteins. Altogether, association of RBPs to Bcl2 AREs contributes to Bcl2 journal.pone.0116899 protein expression by stabilizing Bcl2 mRNA and promotes B cell maintenance. Academic Editor: Carol J Wilusz, Colorado State University, UNITED STATES Received: September 10, 2014 Accepted: December 16, 2014 Introduction Published: February 13, 2015 Human B-cell lymphomas are characterised by sequential genetic alterations that deregulate Copyright: © 2015 Díaz-Muñoz et al. This is an open several pathways, including cell cycle and apoptosis. Genetic translocation affecting proto-on- access article distributed under the terms of the cogenes, such as Bcl2, Bcl6 or c-Myc, are found in many tumours, including follicular B-cell Creative Commons Attribution License, which permits “ ” unrestricted use, distribution, and reproduction in any lymphoma, Burkitt lymphoma and double hit (DH) mature B cell lymphomas [1,2]. In most medium, provided the original author and source are of the cases, genetic juxtaposition of the oncogene to an enhancer of the Ig locus is the cause of credited. increased gene expression. For example, the Bcl2 translocation t(11,14) is commonly found in μ Data Availability Statement: All relevant data are follicular-center B lymphomas. This translocation places the Bcl2 gene under the E enhancer within the paper and its Supporting Information files. of the Igh locus [3]. Enhanced transcription and expression of Bcl2 increases B cell survival [4] and is important for maintenance and progression of tumours [5]. Endogenous expression of Funding: This work was supported by the μ Biotechnology and Biological Sciences Research Bcl2 is not required for the development of E -myc induced B-cell lymphoma, but it is needed Council (http://www.bbsrc.ac.uk/). The funders had no to maintain mature B cells in healthy mice [6,7]. role in study design, data collection and analysis, While gene transcription is anomalous in many tumours, post-transcriptional gene regula- decision to publish, or preparation of the manuscript. tion may remain intact. Chemical modulation of the different post-transcriptional regulatory Competing Interests: The authors have declared mechanisms offers alternative drug-targeting opportunities to reduce oncogene protein expres- that no competing interests exist. sion. RNA molecules are associated with RNA binding proteins (RBPs) during and after their PLOS ONE | DOI:10.1371/journal.pone.0116899 February 13, 2015 1/19 Bcl2 AREs Are Required for Bcl2 Expression transcription. RBPs control RNA splicing, transport, location, stability and translation, modu- lating the nature and content of proteins within the cell. Many mRNA encoding proto- oncogenes are subjected to post-transcriptional regulation, including c-Myc, Bcl6 and Bcl2 [8–10]. Within the 30UTR of these mRNAs, multiple adenine uridine (AU)-rich elements (ARE), including pentamers (AUUUA) and nonamers (UUAUUUAUU), are bound by AU-rich binding proteins (AUBPs), which modulate mRNA stability in a target-dependent manner [11]. Post-transcriptional regulation of Bcl2 mRNA is thought to be a key element for Bcl2 pro- tein expression. The 30UTR of Bcl2 mRNA is considerably longer than the coding sequence and contains several binding motifs for RBPs and microRNAs. In particular, the binding of AUBPs to the AREs present in the proximal region after the stop codon has been functionally implicated in controlling the fate of Bcl2 mRNA (this 300 bp long sequence is described in the manuscript as the Bcl2 ARE-rich sequence). Different biochemical studies suggest that the binding of different AUBPs to Bcl2 AREs exerts opposing effects on Bcl2 mRNA stability. HuR, nucleolin and ErbB3 (Ebp1) may act as mRNA stabilizers, whereas AUF1, Mex3D (Tino) and Tis11b may promote Bcl2 mRNA degradation in vitro [12–16]. Recently, the generation of specific knockout (KO) mice for HuR and AUF1 have shown that both proteins may contrib- ute to Bcl2 mRNA stabilization in B cells [17,18]. Thus, contradictory results have been achieved from in vitro and in vivo studies, and the importance of post-transcriptional regula- tion of Bcl2 mRNA for final protein expression remains unclear. In this study, we have evaluat- ed the impact of the genetic deletion of the Bcl2 ARE-rich sequence on Bcl2 expression in primary B cells. We demonstrate that the binding of RBPs to this sequence of the 3’UTR is di- rectly linked to the stabilization of the Bcl2 mRNA and regulates Bcl2 protein expression with functional consequences for B cell maintenance in vivo. Materials and Methods Generation of Bcl2-AREflox/flox mice Bcl2-AREflox/flox (Bcl2tm1Tnr) conditional mice were generated by gene targeting using standard recombineering technology [19]. The targeting strategy is illustrated in S1A and S1B Fig. Geno- typing of targeted ES cells was performed by Southern blot using the restriction enzyme BsoBI (S1C Fig.). Genotyping of Bcl2-AREflox/flox mice was performed by PCR (primers described in S1 Table). To test for germ-line recombination in Bcl2-AREflox/flox x mb1cre mice, two different qPCR assays were designed: Assay A amplifies the intron 1—exon 2 junction of the Bcl2 gene. Assay B amplifies a region of 100 bp inside the loxP-flanked ARE sequence (S1D Fig.). The copy number and the ratio B/A were calculated as described in the extended materials and methods section (S1 Methods). Ratio B/A = 1 was scored by Bcl2-AREflox/flox x mb1wt (flox/ Δ flox), ratio B/A = 0.5 was scored by Bcl2-AREflox/ x mb1wt mice, which contained one recom- bined allele (flox/Δ). Ratio B/A = 0 was obtained when the Bcl2 ARE sequence was undetect- able in the qPCR assay B (Bcl2-AREflox/flox x mb1cre, called Δ/Δ in the figures) (S1E Fig.). Mouse strains and animal procedures The mouse strains used in this study are: C57BL/6, B6.SJL (B6.SJL-Ptprca Pep3b/BoyJ), FLPe- tg (mice from Dr. F. Stewart backcrossed into the C57BL/6 background), Bcl2-AREflox/flox (Bcl2tm1Tnr), mb1cre (CD79atm1(cre)Reth) and Bcl2.36 (C57BL/6-Tg(BCL2)36Wehi/J). Bone marrow (BM) chimeras were generated using BM cells from B6.SJL, mb1cre and Bcl2- AREflox/flox x mb1cre mice that were harvested and mixed in a 1:1 ratio as follows: Control Δ Δ group—Cells from B6.SJL and mb1cre mice; and ARE / group—Cells from B6.SJL and Bcl2- AREflox/flox x mb1cre mice. The mixture of BM cells was intravenously injected into lethally PLOS ONE | DOI:10.1371/journal.pone.0116899 February 13, 2015 2/19 Bcl2 AREs Are Required for Bcl2 Expression irradiated B6.SJL mice (10 Gy) (3x10^6 cells per mouse). After 8 weeks, cell reconstitution was tested by flow cytometry using specific antibodies against CD19, CD3 and CD45.2 cell surface markers. Peripheral immune tissues were analysed 12 weeks after bone marrow (BM) injection. Flow cytometry Analysis of B and T cell populations in the blood, bone marrow, spleen and peripheral lymph nodes (LNs) was performed using specific antibodies (see S2 Table). DAPI was added to test cell viability. For intracellular staining of Bcl2 protein, the BD Cytofix/Cytoperm Fixation/ Permeabilization Solution Kit (BD Biosciences) was used as indicated by the manufactures. Cell culture and Western blot B cells from inguinal, brachial and axillary lymph nodes, or from spleen, were isolated by nega- tive selection using the B Cell Isolation Kit from Miltenyi Biotec. B cells were cultured in RPMI 1640 Medium (Dutch Modification) (Life Technologies) supplemented with 5% FCS, antibiot- ics, 2 mM L-glutamine, 5 μM β-mercaptoethanol and 1 mM sodium pyruvate. In some experi- ments LPS (10 μg/ml, E. coli O127:B8 Sigma Aldrich) was used for cell stimulation. Total cell extracts were prepared by incubating cells in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS and 0.5% sodium deoxycholate) supplemented with protease inhibitors (Protease inhibitor cocktail 3, Cat. No. p8340, Sigma). After 15 minutes at 4°C, cell extracts were centrifuged and protein concentration in the supernatant was measured using a BCA protein assay (Pierce). 10% polyacrylamide-SDS gels were loaded with the indicat- ed amount of protein extracts (10–20 μg per lane). Proteins were transferred to nitrocellulose membranes, and AUF-1 and tubulin proteins were detected with specific primary antibodies (see S2 Table). Blots were subsequently incubated with specific HRP-conjugated secondary an- tibodies and detected by enhanced chemiluminescence (Amersham Pharmacia Biotech). RNA extraction and qPCR Total RNA from purified cells was isolated using TRIzol (LifeTech). 1 μg of RNA was reverse- transcribed to perform qPCR assays using Platinum Quantitative PCR SuperMix-UDG or SYBR Green PCR Master Mix (Life Technologies).
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