RNA-binding proteins control gene expression and cell fate in the immune system.

RNA-binding proteins (RBPs) are essential for the development and function of the immune system. They interact dynamically with RNA to control its biogenesis and turnover by transcription-dependent and transcription-independent mechanisms. In this Review, we discuss the molecular mechanisms by which RBPs allow gene expression changes to occur at different speeds and to varying degrees, and which RBPs regulate the diversity of the transcriptome and proteome. These proteins are nodes for integration of transcriptional and signaling networks and are intimately linked to intermediary metabolism. They are essential components of regulatory feedback mechanisms that maintain immune tolerance and limit inflammation. The role of RBPs in malignancy and autoimmunity has led to their emergence as targets for the development of new therapeutic modalities.

The RNA-binding protein PTBP1 is necessary for B cell selection in germinal centers.

Antibody affinity maturation occurs in germinal centers (GCs), where B cells cycle between the light zone (LZ) and the dark zone. In the LZ, GC B cells bearing immunoglobulins with the highest affinity for antigen receive positive selection signals from helper T cells, which promotes their rapid proliferation. Here we found that the RNA-binding protein PTBP1 was needed for the progression of GC B cells through late S phase of the cell cycle and for affinity maturation. PTBP1 was required for proper expression of the c-MYC-dependent gene program induced in GC B cells receiving T cell help and directly regulated the alternative splicing and abundance of transcripts that are increased during positive selection to promote proliferation.

The RNA-binding protein HuR is essential for the B cell antibody response.

Post-transcriptional regulation of mRNA by the RNA-binding protein HuR (encoded by Elavl1) is required in B cells for the germinal center reaction and for the production of class-switched antibodies in response to thymus-independent antigens. Transcriptome-wide examination of RNA isoforms and their abundance and translation in HuR-deficient B cells, together with direct measurements of HuR-RNA interactions, revealed that HuR-dependent splicing of mRNA affected hundreds of transcripts, including that encoding dihydrolipoamide S-succinyltransferase (Dlst), a subunit of the 2-oxoglutarate dehydrogenase (α-KGDH) complex. In the absence of HuR, defective mitochondrial metabolism resulted in large amounts of reactive oxygen species and B cell death. Our study shows how post-transcriptional processes control the balance of energy metabolism required for the proliferation and differentiation of B cells.

ORFLine: a bioinformatic pipeline to prioritize small open reading frames identifies candidate secreted small proteins from lymphocytes.

MOTIVATION: The annotation of small open reading frames (smORFs) of <100 codons (<300 nucleotides) is challenging due to the large number of such sequences in the genome. RESULTS: In this study, we developed a computational pipeline, which we have named ORFLine, that stringently identifies smORFs and classifies them according to their position within transcripts. We identified a total of 5744 unique smORFs in datasets from mouse B and T lymphocytes and systematically characterized them using ORFLine. We further searched smORFs for the presence of a signal peptide, which predicted known secreted chemokines as well as novel micropeptides. Four novel micropeptides show evidence of secretion and are therefore candidate mediators of immunoregulatory functions. AVAILABILITY AND IMPLEMENTATION: Freely available on the web at https://github.com/boboppie/ORFLine. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Regulation of Cyclooxygenase-2 Expression in Human T Cells by Glucocorticoid Receptor-Mediated Transrepression of Nuclear Factor of Activated T Cells.

Cyclooxygenase (COX) is the key enzyme in prostanoid synthesis from arachidonic acid (AA). Two isoforms, named COX-1 and COX-2, are expressed in mammalian tissues. The expression of COX-2 isoform is induced by several stimuli including cytokines and mitogens, and this induction is inhibited by glucocorticoids (GCs). We have previously shown that the transcriptional induction of COX-2 occurs early after T cell receptor (TCR) triggering, suggesting functional implications of this enzyme in T cell activation. Here, we show that dexamethasone (Dex) inhibits nuclear factor of activated T cells (NFAT)-mediated COX-2 transcriptional induction upon T cell activation. This effect is dependent on the presence of the GC receptor (GR), but independent of a functional DNA binding domain, as the activation-deficient GRLS7 mutant was as effective as the wild-type GR in the repression of NFAT-dependent transcription. Dex treatment did not disturb NFAT dephosphorylation, but interfered with activation mediated by the N-terminal transactivation domain (TAD) of NFAT, thus pointing to a negative cross-talk between GR and NFAT at the nuclear level. These results unveil the ability of GCs to interfere with NFAT activation and the induction of pro-inflammatory genes such as COX-2, and explain some of their immunomodulatory properties in activated human T cells.

The RNA-binding protein TTP is a global post-transcriptional regulator of feedback control in inflammation.

RNA-binding proteins (RBPs) facilitate post-transcriptional control of eukaryotic gene expression at multiple levels. The RBP tristetraprolin (TTP/Zfp36) is a signal-induced phosphorylated anti-inflammatory protein guiding unstable mRNAs of pro-inflammatory proteins for degradation and preventing translation. Using iCLIP, we have identified numerous mRNA targets bound by wild-type TTP and by a non-MK2-phosphorylatable TTP mutant (TTP-AA) in 1 h LPS-stimulated macrophages and correlated their interaction with TTP to changes at the level of mRNA abundance and translation in a transcriptome-wide manner. The close similarity of the transcriptomes of TTP-deficient and TTP-expressing macrophages upon short LPS stimulation suggested an effective inactivation of TTP by MK2, whereas retained RNA-binding capacity of TTP-AA to 3'UTRs caused profound changes in the transcriptome and translatome, altered NF-κB-activation and induced cell death. Increased TTP binding to the 3'UTR of feedback inhibitor mRNAs, such as Ier3, Dusp1 or Tnfaip3, in the absence of MK2-dependent TTP neutralization resulted in a strong reduction of their protein synthesis contributing to the deregulation of the NF-κB-signaling pathway. Taken together, our study uncovers a role of TTP as a suppressor of feedback inhibitors of inflammation and highlights the importance of fine-tuned TTP activity-regulation by MK2 in order to control the pro-inflammatory response.

Dose-dependent effects of prostaglandin E2 in macrophage adhesion and migration.

Macrophage migration to the focus of infection is a hallmark of the innate immune response. Macrophage spreading, adhesion, and migration through the extracellular matrix require dynamic remodeling of the actin cytoskeleton associated to integrin clustering in podosomes and focal adhesions. Here, we show that prostaglandin E2 (PGE2 ), the main prostaglandin produced by macrophages during inflammation, promote the distinctive dose-dependent formation of podosomes or focal adhesions in macrophages. Low concentrations of PGE2 increased p110γ PI3K expression, phosphorylation of actin-related protein 2, and formation of podosomes, which enhanced macrophage migration in response to chemokines. However, high doses of PGE2 increased phosphorylation of paxillin and focal adhesion kinase, the expression of serine/threonine protein kinase 1, and promoted focal adhesion formation and macrophage adhesion, reducing macrophage chemotaxis. In summary, we describe the dual role of PGE2 as a promoter of macrophage chemotaxis and adhesion, proposing a new model of macrophage migration to the inflammatory focus in the presence of a gradient of PGE2 .

Generation of functionally distinct isoforms of PTBP3 by alternative splicing and translation initiation.

Polypyrimidine tract binding protein (PTBP1) is a widely expressed RNA binding protein that acts as a regulator of alternative splicing and of cytoplasmic mRNA functions. Vertebrates contain two closely-related paralogs with >75% amino acid sequence identity. Early replacement of PTBP1 by PTBP2 during neuronal differentiation causes a concerted set of splicing changes. By comparison, very little is known about the molecular functions or physiological roles of PTBP3, although its expression and conservation throughout the vertebrates suggest a role in haematopoietic cells. To begin to understand its functions we have characterized the mRNA and protein isoform repertoire of PTBP3. Combinatorial alternative splicing events at the 5' end of the gene allow for the generation of eight mRNA and three major protein isoforms. Individual mRNAs generate up to three protein isoforms via alternative translation initiation by re-initiation and leaky scanning using downstream AUG codons. The N-terminally truncated PTBP3 isoforms lack nuclear localization signals and/or most of the RRM1 domain and vary in their RNA binding properties and nuclear/cytoplasmic distribution, suggesting that PTBP3 may have major post-transcriptional cytoplasmic roles. Our findings set the stage for understanding the non-redundant physiological roles of PTBP3.

Cyclooxygenase-2 deficiency in macrophages leads to defective p110γ PI3K signaling and impairs cell adhesion and migration.

Cyclooxygenase (Cox)-2 dependent PGs modulate several functions in many pathophysiological processes, including migration of immune cells. In this study, we addressed the role of Cox-2 in macrophage migration by using in vivo and in vitro models. Upon thioglycolate challenge, CD11b(+) F4/80(+) macrophages showed a diminished ability to migrate to the peritoneal cavity in cox-2(-/-) mice. In vivo migration of cox-2(-/-) macrophages from the peritoneal cavity to lymph nodes, as well as cell adhesion to the mesothelium, was reduced in response to LPS. In vitro migration of cox-2(-/-) macrophages toward MCP-1, RANTES, MIP-1α, or MIP-1ß, as well as cell adhesion to ICAM-1 or fibronectin, was impaired. Defects in cell migration were not due to changes in chemokine receptor expression. Remarkably, cox-2(-/-) macrophages showed a deficiency in focal adhesion formation, with reduced phosphorylation of paxillin (Tyr(188)). Interestingly, expression of the p110γ catalytic subunit of PI3K was severely reduced in the absence of Cox-2, leading to defective Akt phosphorylation, as well as cdc42 and Rac-1 activation. Our results indicate that the paxillin/p110γ-PI3K/Cdc42/Rac1 axis is defective in cox-2(-/-) macrophages, which results in impaired cell adhesion and migration.

Involvement of PGE2 and the cAMP signalling pathway in the up-regulation of COX-2 and mPGES-1 expression in LPS-activated macrophages.

PG (prostaglandin) E2 plays an important role in the modulation of the immune response and the inflammatory process. In the present study, we describe a PGE2 positive feedback for COX (cyclo-oxygenase)-2 and mPGES-1 [microsomal PGES (PGE synthase)-1] expression in the macrophage cell line RAW 264.7. Our results show that PGE2 induces COX-2 and mPGES-1 expression, an effect mimicked by dbcAMP (dibutyryl-cAMP) or forskolin. Furthermore, the cAMP signalling pathway co-operates with LPS (lipopolysaccharide) in the induction of COX-2 and mPGES-1 transcriptional activation. Analysis of the involvement of PGE receptors [EPs (E-prostanoids)] showed that incubation with EP2 agonists up-regulated both COX2 and mPGES-1 mRNA levels. Moreover, EP2 receptor overexpression enhanced the transcriptional activation of COX2 and mPGES-1 promoters. This induction was repressed by the PKA (protein kinase A) inhibitor H89. Activation of the PGE2/EP2/PKA signalling pathway induced the phosphorylation of CREB [CRE (cAMP-response element)-binding protein] in macrophages and stimulated the specific binding of this transcription factor to COX2 and mPGES-1 promoters. Deletion or mutation of potential CRE sites in both promoters diminished their transcriptional activity. In summary, the results of the present study demonstrate that activation of PKA/CREB signalling through the EP2 receptor by PGE2 plays a key role in the expression of COX-2 and mPGES-1 in activated macrophages.

The RNA binding proteins TIA1 and TIAL1 promote Mcl1 mRNA translation to protect germinal center responses from apoptosis.

Germinal centers (GCs) are essential for the establishment of long-lasting antibody responses. GC B cells rely on post-transcriptional RNA mechanisms to translate activation-associated transcriptional programs into functional changes in the cell proteome. However, the critical proteins driving these key mechanisms are still unknown. Here, we show that the RNA binding proteins TIA1 and TIAL1 are required for the generation of long-lasting GC responses. TIA1- and TIAL1-deficient GC B cells fail to undergo antigen-mediated positive selection, expansion and differentiation into B-cell clones producing high-affinity antibodies. Mechanistically, TIA1 and TIAL1 control the transcriptional identity of dark- and light-zone GC B cells and enable timely expression of the prosurvival molecule MCL1. Thus, we demonstrate here that TIA1 and TIAL1 are key players in the post-transcriptional program that selects high-affinity antigen-specific GC B cells.

The RNA regulatory programs that govern lymphocyte development and function.

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 splicing regulators TIA1 and TIAL1 are required for the expression of the DNA damage repair machinery during B cell lymphopoiesis.

B cell lymphopoiesis requires dynamic modulation of the B cell transcriptome for timely coordination of somatic mutagenesis and DNA repair in progenitor B (pro-B) cells. Here, we show that, in pro-B cells, the RNA-binding proteins T cell intracellular antigen 1 (TIA1) and TIA1-like protein (TIAL1) act redundantly to enable developmental progression. They are global splicing regulators that control the expression of hundreds of mRNAs, including those involved in DNA damage repair. Mechanistically, TIA1 and TIAL1 bind to 5' splice sites for exon definition, splicing, and expression of DNA damage sensors, such as Chek2 and Rif1. In their absence, pro-B cells show exacerbated DNA damage, altered P53 expression, and increased cell death. Our study uncovers the importance of tight regulation of RNA splicing by TIA1 and TIAL1 for the expression of integrative transcriptional programs that control DNA damage sensing and repair during B cell development.

Multiomics analysis couples mRNA turnover and translational control of glutamine metabolism to the differentiation of the activated CD4+ T cell.

The ZFP36 family of RNA-binding proteins acts post-transcriptionally to repress translation and promote RNA decay. Studies of genes and pathways regulated by the ZFP36 family in CD4+ T cells have focussed largely on cytokines, but their impact on metabolic reprogramming and differentiation is unclear. Using CD4+ T cells lacking Zfp36 and Zfp36l1, we combined the quantification of mRNA transcription, stability, abundance and translation with crosslinking immunoprecipitation and metabolic profiling to determine how they regulate T cell metabolism and differentiation. Our results suggest that ZFP36 and ZFP36L1 act directly to limit the expression of genes driving anabolic processes by two distinct routes: by targeting transcription factors and by targeting transcripts encoding rate-limiting enzymes. These enzymes span numerous metabolic pathways including glycolysis, one-carbon metabolism and glutaminolysis. Direct binding and repression of transcripts encoding glutamine transporter SLC38A2 correlated with increased cellular glutamine content in ZFP36/ZFP36L1-deficient T cells. Increased conversion of glutamine to α-ketoglutarate in these cells was consistent with direct binding of ZFP36/ZFP36L1 to Gls (encoding glutaminase) and Glud1 (encoding glutamate dehydrogenase). We propose that ZFP36 and ZFP36L1 as well as glutamine and α-ketoglutarate are limiting factors for the acquisition of the cytotoxic CD4+ T cell fate. Our data implicate ZFP36 and ZFP36L1 in limiting glutamine anaplerosis and differentiation of activated CD4+ T cells, likely mediated by direct binding to transcripts of critical genes that drive these processes.

The RNA-binding protein HuR is required for maintenance of the germinal centre response.

The germinal centre (GC) is required for the generation of high affinity antibodies and immunological memory. Here we show that the RNA binding protein HuR has an essential function in GC B cells to sustain the GC response. In its absence, the GC reaction and production of high-affinity antibody is severely impaired. Mechanistically, HuR affects the transcriptome qualitatively and quantitatively. The expression and splicing patterns of hundreds of genes are altered in the absence of HuR. Among these genes, HuR is required for the expression of Myc and a Myc-dependent transcriptional program that controls GC B cell proliferation and Ig somatic hypermutation. Additionally, HuR regulates the splicing and abundance of mRNAs required for entry into and transition through the S phase of the cell cycle, and it modulates a gene signature associated with DNA deamination protecting GC B cells from DNA damage and cell death.

Antigen presentation between T cells drives Th17 polarization under conditions of limiting antigen.

T cells form immunological synapses with professional antigen-presenting cells (APCs) resulting in T cell activation and the acquisition of peptide antigen-MHC (pMHC) complexes from the plasma membrane of the APC. They thus become APCs themselves. We investigate the functional outcome of T-T cell antigen presentation by CD4 T cells and find that the antigen-presenting T cells (Tpres) predominantly differentiate into regulatory T cells (Treg), whereas T cells that have been stimulated by Tpres cells predominantly differentiate into Th17 pro-inflammatory cells. Using mice deficient in pMHC uptake by T cells, we show that T-T antigen presentation is important for the development of experimental autoimmune encephalitis and Th17 cell differentiation in vivo. By varying the professional APC:T cell ratio, we can modulate Treg versus Th17 differentiation in vitro and in vivo, suggesting that T-T antigen presentation underlies proinflammatory responses in conditions of antigen scarcity.

A post-transcriptional program of chemoresistance by AU-rich elements and TTP in quiescent leukemic cells.

BACKGROUND: Quiescence (G0) is a transient, cell cycle-arrested state. By entering G0, cancer cells survive unfavorable conditions such as chemotherapy and cause relapse. While G0 cells have been studied at the transcriptome level, how post-transcriptional regulation contributes to their chemoresistance remains unknown. RESULTS: We induce chemoresistant and G0 leukemic cells by serum starvation or chemotherapy treatment. To study post-transcriptional regulation in G0 leukemic cells, we systematically analyzed their transcriptome, translatome, and proteome. We find that our resistant G0 cells recapitulate gene expression profiles of in vivo chemoresistant leukemic and G0 models. In G0 cells, canonical translation initiation is inhibited; yet we find that inflammatory genes are highly translated, indicating alternative post-transcriptional regulation. Importantly, AU-rich elements (AREs) are significantly enriched in the upregulated G0 translatome and transcriptome. Mechanistically, we find the stress-responsive p38 MAPK-MK2 signaling pathway stabilizes ARE mRNAs by phosphorylation and inactivation of mRNA decay factor, Tristetraprolin (TTP) in G0. This permits expression of ARE mRNAs that promote chemoresistance. Conversely, inhibition of TTP phosphorylation by p38 MAPK inhibitors and non-phosphorylatable TTP mutant decreases ARE-bearing TNFα and DUSP1 mRNAs and sensitizes leukemic cells to chemotherapy. Furthermore, co-inhibiting p38 MAPK and TNFα prior to or along with chemotherapy substantially reduces chemoresistance in primary leukemic cells ex vivo and in vivo. CONCLUSIONS: These studies uncover post-transcriptional regulation underlying chemoresistance in leukemia. Our data reveal the p38 MAPK-MK2-TTP axis as a key regulator of expression of ARE-bearing mRNAs that promote chemoresistance. By disrupting this pathway, we develop an effective combination therapy against chemosurvival.