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B cell development in the bone marrow is normal in Bcl2-AREflox/flox x mb1cre mice. A, Flow Cytometry analysis of pro-B cells (B220+ IgM- IgD- c-kit+ CD25- cells), pre-B cells (B220+ IgM- IgD- c-kit- CD25+ cells), immature-B cells (B220+ CD43- IgM+ IgD- cells) and mature-B cells (B220high CD43- IgM+ IgD+ cells) from the bone marrow of Bcl2-AREflox/flox x mb1wt (flox/flox) and Bcl2-AREflox/flox x mb1cre (∆/∆) mice. N = 6–7 mice per genotype. The mean percentage of cells in each gate is shown within the plots. B, Quantification of total number of pro-, pre-, immature- and mature-B cells. A Mann-Whitney test was performed for statistical analysis of the data. P values are shown for each cell population.
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Post-transcriptional mRNA regulation by RNA binding proteins (RBPs) associated with AU-rich elements (AREs) present in the 3' untranslated region (3'UTR) of specific mRNAs modulates 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 m...
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... BCL2 is a key antiapoptotic factor expressed in immature and memory B cells that is down-regulated in B cells during the germinal centre reaction (Peperzak et al. 2017;Shen et al. 2004). hnRNP D (AUF1) appears to promote expression of BCL2 and BCL-XL to maintain appropriate proportions of different B cell subsets, such as follicular B cells (Díaz-Muñoz et al. 2015;Sadri et al. 2010). AUF1 specifically binds to BCL2 AU-rich elements while hnRNP L binds CA elements in the 3'UTR to stabilize the mRNA and prevent decay (Lim et al. 2010, p. 7). ...
Heterogeneous nuclear ribonucleoproteins (hnRNPs) are among the most abundantly expressed RNA binding proteins in the cell and play major roles in all facets of RNA metabolism. hnRNPs are increasingly appreciated as essential for mammalian B cell development by regulating the carefully ordered expression of specific genes. Due to this tight regulation of the hnRNP-RNA network, it is no surprise that a growing number of genes encoding hnRNPs have been causally associated with the onset or progression of many cancers, including B cell neoplasms. Here we discuss our current understanding of hnRNP-driven regulation in normal, perturbed, and malignant B cells, and the most recent and emerging therapeutic innovations aimed at targeting the hnRNP–RNA network in lymphoma.
... Moreover, long 3'UTRs can form secondary structures (stemloop) with stabilizing effects, which perhaps short 3'UTRs do not form. Furthermore, APA events and the diversity of regulatory factors may vary according to cell type or state, tissue type or even with environmental stimulus, as well as the availability and performance of trans-regulatory factors (Deridder et al., 2012;Ulitsky et al., 2012;Lackford et al., 2014;Dıáz-muñoz et al., 2015;Kim et al., 2016). ...
... Site-directed mutagenesis of ARE present in 3'UTRs from her1 was enough to prolong the halflife of reporter mRNAs (Tietz et al., 2020). On the other hand, deletion of a sequence in the 3'UTR of BCL2 with a high content of AREs promoted instability, resulting in low BCL2 levels in murine B cells (Dıáz-muñoz et al., 2015). Interestingly, ARE has the potential to induce exosomal degradation of mRNAs without the need for mediation of other proteins, since RNase domains present in exosomal complex proteins have high affinity for AUrich sequences (Anderson et al., 2006). ...
Molecular biotechnology has made it possible to explore the potential of plants for different purposes. The 3’ regulatory regions have a great diversity of cis-regulatory elements directly involved in polyadenylation, stability, transport and mRNA translation, essential to achieve the desired levels of gene expression. A complex interaction between the cleavage and polyadenylation molecular complex and cis-elements determine the polyadenylation site, which may result in the choice of non-canonical sites, resulting in alternative polyadenylation events, involved in the regulation of more than 80% of the genes expressed in plants. In addition, after transcription, a wide array of RNA-binding proteins interacts with cis-acting elements located mainly in the 3’ untranslated region, determining the fate of mRNAs in eukaryotic cells. Although a small number of 3’ regulatory regions have been identified and validated so far, many studies have shown that plant 3’ regulatory regions have a higher potential to regulate gene expression in plants compared to widely used 3’ regulatory regions, such as NOS and OCS from Agrobacterium tumefaciens and 35S from cauliflower mosaic virus. In this review, we discuss the role of 3’ regulatory regions in gene expression, and the superior potential that plant 3’ regulatory regions have compared to NOS, OCS and 35S 3’ regulatory regions.
... hnRNPC regulates Bcl2 mRNA decay through the binding to AU-rich regulatory elements (AREs) present in the Bcl2 3'UTR. Gene targeting deletion of these Bcl2 AREs diminishes Bcl2 mRNA stability and protein levels in primary B cells, decreasing life-span of transitional (T) and FO B cells (149). Stringent regulation of mRNA abundance is also essential for the maintenance of marginal zone precursors (MZP) and mature marginal zone (MZ) B cells. ...
Fighting external pathogens requires an ever-changing immune system that relies on tight regulation of gene expression. Transcriptional control is the first step to build efficient responses while preventing immunodeficiencies and autoimmunity. Post-transcriptional regulation of RNA editing, location, stability, and translation are the other key steps for final gene expression, and they are all controlled by RNA-binding proteins (RBPs). Nowadays we have a deep understanding of how transcription factors control the immune system but recent evidences suggest that post-transcriptional regulation by RBPs is equally important for both development and activation of immune responses. Here, we review current knowledge about how post-transcriptional control by RBPs shapes our immune system and discuss the perspective of RBPs being the key players of a hidden immune cell epitranscriptome.
... Ethanol precipitation procedure was follow for mRNA precipitation. mRNA was used for either 3′ RNA-seq library preparation (described below) or it was converted into cDNA to perform RT-qPCR analysis as follows 69 : Briefly, RNA retro-transcription (RT) was performed using SuperScript II (Life Technologies) and qPCR assays were performed using Platinum Quantitative PCR SuperMix-UDG or SYBR Green PCR Master Mix (Life Technologies). Total RNA extraction was performed using TriZol (Life Technologies) and converted into cDNA similarly. ...
... RNA immunoprecipitation (RIP). Validation of Tia1 and Tial1 interaction with selected mRNA targets was performed by RIP as follows 69 : Briefly, RIP assays were performed with cytoplasmic cell extracts were obtained using cytoplasmic cell lysis buffer (10 mM TrisHCl; pH 7.4, 10 mM NaCl, 2.5 mM MgCl 2, 40 µg ml −1 digitonin from Sigma Aldrich) containing protease inhibitors (p8340, Sigma Aldrich) and RNase inhibitors (40 U ml −1 of RNase Out, Life Technologies). After incubation on ice for 15 min, 10 µl of 10% NP-40 were added. ...
Post-transcriptional regulation of cellular mRNA is essential for protein synthesis. Here we describe the importance of mRNA translational repression and mRNA subcellular location for protein expression during B lymphocyte activation and the DNA damage response. Cytoplasmic RNA granules are formed upon cell activation with mitogens, including stress granules that contain the RNA binding protein Tia1. Tia1 binds to a subset of transcripts involved in cell stress, including p53 mRNA, and controls translational silencing and RNA granule localization. DNA damage promotes mRNA relocation and translation in part due to dissociation of Tia1 from its mRNA targets. Upon DNA damage, p53 mRNA is released from stress granules and associates with polyribosomes to increase protein synthesis in a CAP-independent manner. Global analysis of cellular mRNA abundance and translation indicates that this is an extended ATM-dependent mechanism to increase protein expression of key modulators of the DNA damage response.
... For example, the RBP HuR binds to important transcription factors that control the GC reaction and terminal differentiation into antibody-producing cells (Fig. 2a). The importance of these RNA-protein interactions in B cell development and differentiation can be then explored by conditional targeting and deletion of these binding sites as shown for Tnf and Bcl2 [31,32]. 19. ...
Posttranscriptional regulation of gene expression shapes the B cell transcriptome and controls messenger RNA (mRNA) translation into protein. Recent reports have highlighted the importance of RNA binding proteins (RBPs) for mRNA splicing, subcellular location, stability, and translation during B lymphocyte development, activation, and differentiation. Here we describe individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) in primary lymphocytes, a method that maps RNA–protein interactions in a genome-wide scale allowing mechanistic analysis of RBP function. We discuss the latest improvements in iCLIP technology and provide some examples of how integration of the RNA–protein interactome with other high-throughput mRNA sequencing methodologies uncovers the important role of RBP-mediated RNA regulation in key biological cell processes.
... The prosurvival protein Bcl2 is required to maintain mature B cells in healthy mice [77,78]. AUF1 has been shown to bind to the AU-rich sequence in the 3 0 UTR of Bcl2 mRNA in mature primary B cells [79]. Deletion of these AU-rich elements reduces Bcl2 protein expression [79]. ...
... AUF1 has been shown to bind to the AU-rich sequence in the 3 0 UTR of Bcl2 mRNA in mature primary B cells [79]. Deletion of these AU-rich elements reduces Bcl2 protein expression [79]. This is due in part to the lack of AUF1 binding to the 3 0 UTR of Bcl2, which is required for the stabilization and subsequent translation of the Bcl2 transcript, promoting survival of mature B cells [79]. ...
... Deletion of these AU-rich elements reduces Bcl2 protein expression [79]. This is due in part to the lack of AUF1 binding to the 3 0 UTR of Bcl2, which is required for the stabilization and subsequent translation of the Bcl2 transcript, promoting survival of mature B cells [79]. ...
Sequence-specific RNA binding proteins (RBP) are important regulators of the immune response. RBP modulate gene expression by regulating splicing, polyadenylation, localization, translation and decay of target mRNAs. Increasing evidence suggests that RBP play critical roles in the development, activation and function of lymphocyte populations in the immune system. This review will discuss the post-transcriptional regulation of gene expression by RBP during lymphocyte development, with particular focus on the Tristetraprolin family of RBP.
... Furthermore, some miRNA have also been demonstrated to be involved in Bcl-2 post-transcriptional control and in its alterations in human leukemias [35][36][37][38] . Very recently, Díaz-Muñoz MD et al. have disclosed the ability of Bcl-2 AUBP/ARE association to stabilize in vivo Bcl-2 mRNA, contributing to Bcl-2 protein over-production and B cell survival [39] . ...
The Bcl-2 (B-cell lymphoma 2) antiapoptotic gene has been discovered in virtue of its over-expression occurring in B-cell leukemias/lymphomas carrying the 14;18 chromosomal translocation [t(14;18)], which places the Bcl-2 gene next to the immunoglobulin heavy chain (IgH) locus. In this condition, the transcription of the Bcl-2 moiety of the Bcl-2/IgH fusion gene is driven by the four enhancers located in 3’ of the IgH moiety and is, therefore, excessive. This leads to overproduction of Bcl-2 protein, which confers a survival advantage that contributes to neoplastic transformation. Nevertheless, in most malignancies, comprising chronic lymphocytic leukemias, breast, prostate, colorectal and lung cancer, the over-expression of Bcl-2 does not imply chromosomal rearrangements, suggesting that alterations at post-transcriptional level could be involved. Collaborating with the group of Angelo Nicolin (University of Milan, Italy), we first disclosed the existence of a Bcl-2 post-transcriptional control based on interplay among an Adenine and uracil-Rich cis-acting Element (ARE) located in the 3’UTR of Bcl-2 mRNA and several trans-acting ARE-Binding Proteins (AUBPs). We also demonstrated its deregulation in human leukemias/lymphomas. In particular, we have identified some Bcl-2 AUBPs - such as AUF-1, TINO/hMex-3D, the Bcl-2 protein itself and ζ-Crystallin - and described their qualitative or quantitative alterations in cancer cells. Moreover, in the attempt to correct Bcl-2 deregulation in the human diseases characterized by defects or excesses of apoptosis, we have modulated exogenously Bcl-2 expression by means of different antisense strategies. In this research highlight, we briefly report our proceedings, in which a long non-coding Bcl-2/IgH antisense RNA (Bcl-2/IgH AS) we discovered in a serendipitous manner has played a key role.
Lymphocytes require of constant and dynamic changes in their transcriptome for timely activation and production of effector molecules to combat external pathogens. Synthesis and translation of messenger (m)RNAs into these effector proteins is controlled both quantitatively and qualitatively by RNA binding proteins (RBPs). RBP‐dependent regulation of RNA editing, subcellular location, stability, and translation shapes immune cell development and immunity. Extensive evidences have now been gathered from few model RBPs, HuR, PTBP1, ZFP36, and Roquin. However, recently developed methodologies for global characterization of protein:RNA interactions suggest the existence of complex RNA regulatory networks in which RBPs co‐ordinately regulate the fate of sets of RNAs controlling cellular pathways and functions. In turn, RNA can also act as scaffolding of functionally related proteins modulating their activation and function. Here we review current knowledge about how RBP‐dependent regulation of RNA shapes our immune system and discuss about the existence of a hidden immune cell epitranscriptome.
This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein‐RNA Interactions: Functional Implications
The human nonsense-mediated mRNA decay pathway (NMD) performs quality control and regulatory functions within complex post-transcriptional regulatory networks. In addition to degradation-promoting factors, efficient and accurate detection of NMD substrates involves proteins that safeguard normal mRNAs. Here, we identify hnRNP L as a factor that protects mRNAs with NMD-inducing features including long 3′UTRs. Using biochemical and transcriptome-wide approaches, we provide evidence that the susceptibility of a given transcript to NMD can be modulated by its 3′UTR length and ability to recruit hnRNP L. Integrating these findings with the previously defined role of polypyrimidine tract binding protein 1 in NMD evasion enables enhanced prediction of transcript susceptibility to NMD. Unexpectedly, this system is subverted in B cell lymphomas harboring translocations that produce BCL2:IGH fusion mRNAs. CRISPR/Cas9 deletion of hnRNP L binding sites near the BCL2 stop codon reduces expression of the fusion mRNAs and induces apoptosis. Together, our data indicate that protection by hnRNP L overrides the presence of multiple 3′UTR introns, allowing these aberrant mRNAs to evade NMD and promoting BCL2 overexpression and neoplasia. Published 2018. This article is a U.S. Government work and is in the public domain in the USA
BCL-2-related ovarian killer (BOK) is a pro-apoptotic BAX-like member of the BCL-2 family with suggested tumor suppressor activity. The molecular mechanisms regulating BOK expression are poorly understood and fail to explain a frequent lack of concordance between protein and transcript levels. Here, we describe a potent post-transcriptional mechanism that negatively regulates BOK expression mediated by conserved (AU/U)-rich elements within its 3’ UTR. Using proteomics approaches we identified TRIM28 as a key component associating with U-rich elements in the human BOK 3’ UTR, resulting in a dramatic reduction of BOK expression. TRIM28 is overexpressed in several cancers, correlating with poor patient outcome, whereas the BOK locus is frequently deleted or its expression downregulated in human cancers. Data mining indicated that, for certain cancers, high TRIM28 and low BOK expression are significantly correlated in the stratum of patients with the worst survival, suggesting that this mechanism might be of potential therapeutic value.
