The thymocyte-specific RNA-binding protein Arpp21 provides TCR repertoire diversity by binding to the 3'-UTR and promoting Rag1 mRNA expression.

The regulation of thymocyte development by RNA-binding proteins (RBPs) is largely unexplored. We identify 642 RBPs in the thymus and focus on Arpp21, which shows selective and dynamic expression in early thymocytes. Arpp21 is downregulated in response to T cell receptor (TCR) and Ca2+ signals. Downregulation requires Stim1/Stim2 and CaMK4 expression and involves Arpp21 protein phosphorylation, polyubiquitination and proteasomal degradation. Arpp21 directly binds RNA through its R3H domain, with a preference for uridine-rich motifs, promoting the expression of target mRNAs. Analysis of the Arpp21-bound transcriptome reveals strong interactions with the Rag1 3'-UTR. Arpp21-deficient thymocytes show reduced Rag1 expression, delayed TCR rearrangement and a less diverse TCR repertoire. This phenotype is recapitulated in Rag1 3'-UTR mutant mice harboring a deletion of the Arpp21 response region. These findings show how thymocyte-specific Arpp21 promotes Rag1 expression to enable TCR repertoire diversity until signals from the TCR terminate Arpp21 and Rag1 activities.

Unrestrained cleavage of Roquin-1 by MALT1 induces spontaneous T cell activation and the development of autoimmunity.

Constitutive activation of the MALT1 paracaspase in conventional T cells of Malt1TBM/TBM (TRAF6 Binding Mutant = TBM) mice causes fatal inflammation and autoimmunity, but the involved targets and underlying molecular mechanisms are unknown. We genetically rendered a single MALT1 substrate, the RNA-binding protein (RBP) Roquin-1, insensitive to MALT1 cleavage. These Rc3h1Mins/Mins mice showed normal immune homeostasis. Combining Rc3h1Mins/Mins alleles with those encoding for constitutively active MALT1 (TBM) prevented spontaneous T cell activation and restored viability of Malt1TBM/TBM mice. Mechanistically, we show how antigen/MHC recognition is translated by MALT1 into Roquin cleavage and derepression of Roquin targets. Increasing T cell receptor (TCR) signals inactivated Roquin more effectively, and only high TCR strength enabled derepression of high-affinity targets to promote Th17 differentiation. Induction of experimental autoimmune encephalomyelitis (EAE) revealed increased cleavage of Roquin-1 in disease-associated Th17 compared to Th1 cells in the CNS. T cells from Rc3h1Mins/Mins mice did not efficiently induce the high-affinity Roquin-1 target IκBNS in response to TCR stimulation, showed reduced Th17 differentiation, and Rc3h1Mins/Mins mice were protected from EAE. These data demonstrate how TCR signaling and MALT1 activation utilize graded cleavage of Roquin to differentially regulate target mRNAs that control T cell activation and differentiation as well as the development of autoimmunity.

Characterising a homozygous two-exon deletion in UQCRH: comparing human and mouse phenotypes.

Mitochondrial disorders are clinically and genetically diverse, with isolated complex III (CIII) deficiency being relatively rare. Here, we describe two affected cousins, presenting with recurrent episodes of severe lactic acidosis, hyperammonaemia, hypoglycaemia and encephalopathy. Genetic investigations in both cases identified a homozygous deletion of exons 2 and 3 of UQCRH, which encodes a structural complex III (CIII) subunit. We generated a mouse model with the equivalent homozygous Uqcrh deletion (Uqcrh-/- ), which also presented with lactic acidosis and hyperammonaemia, but had a more severe, non-episodic phenotype, resulting in failure to thrive and early death. The biochemical phenotypes observed in patient and Uqcrh-/- mouse tissues were remarkably similar, displaying impaired CIII activity, decreased molecular weight of fully assembled holoenzyme and an increase of an unexpected large supercomplex (SXL ), comprising mostly of one complex I (CI) dimer and one CIII dimer. This phenotypic similarity along with lentiviral rescue experiments in patient fibroblasts verifies the pathogenicity of the shared genetic defect, demonstrating that the Uqcrh-/- mouse is a valuable model for future studies of human CIII deficiency.

Defining the RBPome of primary T helper cells to elucidate higher-order Roquin-mediated mRNA regulation.

Post-transcriptional gene regulation in T cells is dynamic and complex as targeted transcripts respond to various factors. This is evident for the Icos mRNA encoding an essential costimulatory receptor that is regulated by several RNA-binding proteins (RBP), including Roquin-1 and Roquin-2. Here, we identify a core RBPome of 798 mouse and 801 human T cell proteins by utilizing global RNA interactome capture (RNA-IC) and orthogonal organic phase separation (OOPS). The RBPome includes Stat1, Stat4 and Vav1 proteins suggesting unexpected functions for these transcription factors and signal transducers. Based on proximity to Roquin-1, we select ~50 RBPs for testing coregulation of Roquin-1/2 targets by induced expression in wild-type or Roquin-1/2-deficient T cells. Besides Roquin-independent contributions from Rbms1 and Cpeb4 we also show Roquin-1/2-dependent and target-specific coregulation of Icos by Celf1 and Igf2bp3. Connecting the cellular RBPome in a post-transcriptional context, we find contributions from multiple RBPs to the prototypic regulation of mRNA targets by individual trans-acting factors.

Posttranscriptional regulation of T helper cell fate decisions.

T helper cell subsets orchestrate context- and pathogen-specific responses of the immune system. They mostly do so by secreting specific cytokines that attract or induce activation and differentiation of other immune or nonimmune cells. The differentiation of T helper 1 (Th1), Th2, T follicular helper, Th17, and induced regulatory T cell subsets from naive T cells depends on the activation of intracellular signal transduction cascades. These cascades originate from T cell receptor and costimulatory receptor engagement and also receive critical input from cytokine receptors that sample the cytokine milieu within secondary lymphoid organs. Signal transduction then leads to the expression of subset-specifying transcription factors that, in concert with other transcription factors, up-regulate downstream signature genes. Although regulation of transcription is important, recent research has shown that posttranscriptional and posttranslational regulation can critically shape or even determine the outcome of Th cell differentiation. In this review, we describe how specific microRNAs, long noncoding RNAs, RNA-binding proteins, and ubiquitin-modifying enzymes regulate their targets to skew cell fate decisions.

Induced miR-99a expression represses Mtor cooperatively with miR-150 to promote regulatory T-cell differentiation.

Peripheral induction of regulatory T (Treg) cells provides essential protection from inappropriate immune responses. CD4(+) T cells that lack endogenous miRNAs are impaired to differentiate into Treg cells, but the relevant miRNAs are unknown. We performed an overexpression screen with T-cell-expressed miRNAs in naive mouse CD4(+) T cells undergoing Treg differentiation. Among 130 candidates, the screen identified 29 miRNAs with a negative and 10 miRNAs with a positive effect. Testing reciprocal Th17 differentiation revealed specific functions for miR-100, miR-99a and miR-10b, since all of these promoted the Treg and inhibited the Th17 program without impacting on viability, proliferation and activation. miR-99a cooperated with miR-150 to repress the expression of the Th17-promoting factor mTOR. The comparably low expression of miR-99a was strongly increased by the Treg cell inducer "retinoic acid", and the abundantly expressed miR-150 could only repress Mtor in the presence of miR-99a. Our data suggest that induction of Treg cell differentiation is regulated by a miRNA network, which involves cooperation of constitutively expressed as well as inducible miRNAs.

Degradation of oligouridylated histone mRNAs: see UUUUU and goodbye.

During the cell cycle the expression of replication-dependent histones is tightly coupled to DNA synthesis. Histone messenger RNA (mRNA) levels strongly increase during early S-phase and rapidly decrease at the end of it. Here, we review the degradation of replication-dependent histone mRNAs, a paradigm of post-transcriptional gene regulation, in the context of processing, translation, and oligouridylation. Replication-dependent histone transcripts are characterized by the absence of introns and by the presence of a stem-loop structure at the 3' end of a very short 3' untranslated region (UTR). These features, together with a need for active translation, are a prerequisite for their rapid decay. The degradation is induced by 3' end additions of untemplated uridines, performed by terminal uridyl transferases. Such 3' oligouridylated transcripts are preferentially bound by the heteroheptameric LSM1-7 complex, which also interacts with the 3'→5' exonuclease ERI1 (also called 3'hExo). Presumably in cooperation with LSM1-7 and aided by the helicase UPF1, ERI1 degrades through the stem-loop of oligouridylated histone mRNAs in repeated rounds of partial degradation and reoligouridylation. Although histone mRNA decay is now known in some detail, important questions remain open: How is ceasing nuclear DNA replication relayed to the cytoplasmic histone mRNA degradation? Why is translation important for this process? Recent research on factors such as SLIP1, DBP5, eIF3, CTIF, CBP80/20, and ERI1 has provided new insights into the 3' end formation, the nuclear export, and the translation of histone mRNAs. We discuss how these results fit with the preparation of histone mRNAs for degradation, which starts as early as these transcripts are generated.

Roquin paralogs 1 and 2 redundantly repress the Icos and Ox40 costimulator mRNAs and control follicular helper T cell differentiation.

The Roquin-1 protein binds to messenger RNAs (mRNAs) and regulates gene expression posttranscriptionally. A single point mutation in Roquin-1, but not gene ablation, increases follicular helper T (Tfh) cell numbers and causes lupus-like autoimmune disease in mice. In T cells, we did not identify a unique role for the much lower expressed paralog Roquin-2. However, combined ablation of both genes induced accumulation of T cells with an effector and follicular helper phenotype. We showed that Roquin-1 and Roquin-2 proteins redundantly repressed the mRNA of inducible costimulator (Icos) and identified the Ox40 costimulatory receptor as another shared mRNA target. Combined acute deletion increased Ox40 signaling, as well as Irf4 expression, and imposed Tfh differentiation on CD4(+) T cells. These data imply that both proteins maintain tolerance by preventing inappropriate T cell activation and Tfh cell differentiation, and that Roquin-2 compensates in the absence of Roquin-1, but not in the presence of its mutated form.

In human glioblastomas transcript elongation by alternative polyadenylation and miRNA targeting is a potent mechanism of MGMT silencing.

Favorable outcome after chemotherapy of glioblastomas cannot unequivocally be linked to promoter hypermethylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene encoding a DNA repair enzyme associated with resistance to alkylating agents. This indicates that molecular mechanisms determining MGMT expression have not yet been fully elucidated. We here show that glioblastomas are capable to downregulate MGMT expression independently of promoter methylation by elongation of the 3'-UTR of the mRNA, rendering the alternatively polyadenylated transcript susceptible to miRNA-mediated suppression. While the elongated transcript is poorly expressed in normal brain, its abundance in human glioblastoma specimens is inversely correlated with MGMT mRNA expression. Using a bioinformatically guided experimental approach, we identified miR-181d, miR-767-3p, and miR-648 as significant post-transcriptional regulators of MGMT in glioblastomas; the first two miRNAs induce MGMT mRNA degradation, the latter affects MGMT protein translation. A regression model including the two miRNAs influencing MGMT mRNA expression and the MGMT methylation status reliably predicts The Cancer Genome Atlas MGMT expression data. Responsivity of MGMT expressing T98G glioma cells to temozolomide was significantly enhanced after transfection of miR-181d, miR-767-3p, and miR-648. Taken together, our results uncovered alternative polyadenylation of the MGMT 3'-UTR and miRNA targeting as new mechanisms of MGMT silencing.

Eri1 degrades the stem-loop of oligouridylated histone mRNAs to induce replication-dependent decay.

The exoRNase Eri1 inhibits RNA interference and trims the 5.8S rRNA 3' end. It also binds to the stem-loop of histone mRNAs, but the functional importance of this interaction remains elusive. Histone mRNAs are normally degraded at the end of S phase or after pharmacological inhibition of replication. Both processes are impaired in Eri1-deficient mouse cells, which instead accumulate oligouridylated histone mRNAs. Eri1 trims the mature histone mRNAs by two unpaired nucleotides at the 3' end but stalls close to the double-stranded stem. Upon oligouridylation of the histone mRNA, the Lsm1-7 heteroheptamer recognizes the oligo(U) tail and interacts with Eri1, whose catalytic activity is then able to degrade the stem-loop in a stepwise manner. These data demonstrate how degradation of histone mRNAs is initiated when 3' oligouridylation creates a cis element that enables Eri1 to process the double-stranded stem-loop structure.

Measuring microRNA expression in size-limited FACS-sorted and microdissected samples.

MicroRNAs (miRNAs) are small noncoding RNAs of an average length of 22 nucleotides, which repress translation of a large number of target mRNAs. The particular importance of this group of small RNAs arises from the ever growing evidence that they control many biological processes, such as differentiation, proliferation, and apoptosis and that deregulation of individual miRNAs frequently results in cancer. The expression of miRNAs is spatially and temporarily fine-tuned and expression levels can reach more than 50,000 copies of one miRNA within a single cell. It is well documented that the comparison of miRNA signatures of normal and diseased tissues results in a small number of differentially expressed miRNAs, which are consequently of high diagnostic value. However, measuring miRNA expression can easily produce false-positive results, due to the high sequence similarity of the miRNAs within families and because biologically inactive pre-miRNAs as well as contaminating bystander cells may falsify the signal. The application of a quantitative PCR-based method is described here to specifically and reliably detect miRNA expression levels from as little as 50 cells. Pure cell populations were either derived from fluorescence-activated cell sorting (FACS) or laser capture microdissection (LCM). Importantly, a combination of quantitative PCR and LCM can also be applied to measure miRNA expression of cells obtained from formalin-fixed, paraffin-embedded (FFPE) tissues, thereby giving experimental access to archives with large numbers of routinely collected normal and diseased tissue samples.

Roquin binds inducible costimulator mRNA and effectors of mRNA decay to induce microRNA-independent post-transcriptional repression.

The molecular mechanism by which roquin controls the expression of inducible costimulator (ICOS) to prevent autoimmunity remains unsolved. Here we show that in helper T cells, roquin localized to processing (P) bodies and downregulated ICOS expression. The repression was dependent on the RNA helicase Rck, and roquin interacted with Rck and the enhancer of decapping Edc4, which act together in mRNA decapping. Sequences in roquin that confer P-body localization were essential for roquin-mediated ICOS repression. However, this process did not require microRNAs or the RNA-induced silencing complex (RISC). Instead, roquin bound ICOS mRNA directly, showing an intrinsic preference for a previously unrecognized sequence in the 3' untranslated region (3' UTR). Our results support a model in which roquin controls ICOS expression through binding to the 3' UTR of ICOS mRNA and by interacting with proteins that confer post-transcriptional repression.

Interleukin-9 (IL-9) and NPM-ALK each generate mast cell hyperplasia as single 'hit' and cooperate in producing a mastocytosis-like disease in mice.

Mast cell neoplasms are characterized by abnormal growth and focal accumulation of mast cells (MC) in one or more organs. Although several cytokines, including stem cell factor (SCF) and interleukin-9 (IL-9) have been implicated in growth of normal MC, little is known about pro-oncogenic molecules and conditions triggering differentiation and growth of MC far enough to lead to the histopathological picture of overt mastocytosis. The anaplastic lymphoma kinase (ALK) has recently been implicated in growth of neoplastic cells in malignant lymphomas. Here, we describe that transplantation of NPM-ALK-transplanted mouse bone marrow progenitors into lethally irradiated IL-9 transgenic mice not only results in lymphoma-formation, but also in the development of a neoplastic disease exhibiting histopathological features of systemic mastocytosis, including multifocal dense MC-infiltrates, occasionally with devastating growth in visceral organs. Transplantation of NPM-ALK-transduced progenitors into normal mice or maintenance of IL-9-transgenic mice without NPM-ALK each resulted in MC hyperplasia, but not in mastocytosis. Neoplastic MC in mice not only displayed IL-9, but also the IL-9 receptor, and the same was found to hold true for human neoplastic MC. Together, our data show that neoplastic MC express IL-9 receptors, that IL-9 and NPM-ALK upregulate MC-production in vivo, and that both'hits' act in concert to induce a mastocytosis-like disease in mice. These data may have pathogenetic and clinical implications and fit well with the observation that neoplastic MC in advanced SM strongly express NPM and multiple "lymphoid" antigens including CD25 and CD30.

A robust methodology to study urine microRNA as tumor marker: microRNA-126 and microRNA-182 are related to urinary bladder cancer.

OBJECTIVES: MicroRNAs have been shown to be related to specific types of malignant cell growth. In case of urothelial bladder cancer (BCa), novel noninvasive diagnosis is particularly required and it is attractive to consider, as urine is an easily available source for molecular markers including RNA. In this context, we aimed to develop a clinically applicable and sensitive protocol for the preparation and molecular analysis of low molecular weight RNA from urine samples obtained from bladder cancer patients or healthy volunteers. MATERIALS AND METHODS: First, a method was developed for the preparation of low molecular weight RNA from a set of urine samples from different donor groups: (1) patients with low-grade BCa, (2) patients with high-grade BCa, (3) patients with urinary tract infections, (4) healthy donors; each n = 9. The RNA extracts were then used to monitor a number of 157 microRNA species by quantitative reverse transcriptase-polymerase chain reaction. Subsequently, those microRNAs that showed a higher abundance in urine samples from BCa patients were detected in an independent set of urine samples (n = 47). RESULTS: The significance and diagnostic usefulness of this methodology is reflected by the finding that the RNA ratio of microRNA-126:microRNA-152 enabled the detection of BCa from urine at a specificity of 82% and a sensitivity of 72%, with an area under the curve of 0.768 (95% confidence interval, 0.605-0.931). CONCLUSIONS: This study describes a novel, robust, and useful technology platform that is suitable to analyze small RNAs, including novel RNA-based tumor markers, in urine samples. A detailed technical analysis of this methodology provides new insights into the characteristics of urine microRNA such as composition and the donor-dependent variability.

MicroRNA signatures characterize diffuse large B-cell lymphomas and follicular lymphomas.

MicroRNAs (miRNA, miR) are negative regulators of gene expression that play an important role in diverse biological processes such as development, cell growth, apoptosis and haematopoiesis, suggesting their association with cancer. Here we analysed the expression signatures of 157 miRNAs in 58 diffuse large B-cell lymphoma (DLBCL), 46 follicular lymphoma (FL) and seven non-neoplastic lymph nodes (LN). Comparison of the possible combinations of DLBCL-, FL- and LN resulted in specific DLBCL- and FL-signatures, which include miRNAs with previously published function in haematopoiesis (MIRN150 and MIRN155) or tumour development (MIRN210, MIRN10A, MIRN17-5P and MIRN145). As compared to LN, some miRNAs are differentially regulated in both lymphoma types (MIRN155, MIRN210, MIRN106A, MIRN149 and MIRN139). Conversely, some miRNAs show lymphoma-specific aberrant expression, such as MIRN9/9*, MIRN301, MIRN338 and MIRN213 in FL and MIRN150, MIRN17-5P, MIRN145, MIRN328 and others in DLBCL. A classification tree was computed using four miRNAs (MIRN330, MIRN17-5P, MIRN106a and MIRN210) to correctly identify 98% of all 111 cases that were analysed in this study. Finally, eight miRNAs were found to correlate with event-free and overall survival in DLBCL including known tumour suppressors (MIRN21, MIRN127 and MIRN34a) and oncogenes (MIRN195 and MIRNLET7G).

MicroRNAs grow up in the immune system.

MicroRNA (miRNA) target predictions support a view in which each miRNA regulates translation and stability of several hundred messenger RNAs (mRNAs). Studies that overexpress individual miRNAs typically uncover relative subtle inhibition of the predicted targets. Accordingly, most miRNAs expressed in a given cell type may serve the function to broadly inhibit cell-type-inappropriate gene expression and deepen a pre-existing differentiation program. However, recent functional analyses of miRNAs in the immune system reveal that many cellular decisions are controlled by single miRNAs that entail significant downregulation of one or few target proteins. Investigations of these miRNA/mRNA pairs showed that miRNA-adjusted target protein levels are crucial at specific cellular transition points. Here, we will review recent advances in the regulation of the miRNA pathway and discuss how miRNAs control immune functions.

Unlocking pathology archives for microRNA-profiling.

BACKGROUND: MicroRNAs (miRNAs) are approximately 22 nucleotide long, non-coding RNAs that regulate gene expression by binding to the 3'-untranslated region of target mRNAs and also a variety of cellular processes. It has recently been established that dysregulation of miRNA expression can be detected in the majority of human cancers. A variety of high-throughput screening methods has been developed to identify dysregulated miRNA species in tumours. For retrospective clinical studies formalin-fixed, paraffin-embedded (FFPE) tissue is the most widely used material. MATERIALS AND METHODS: The miRNA expression profiles of freshly frozen (CRYO) and FFPE tissues of seven tonsil and four liver samples were compared, using a qPCR-based assay, profiling 157 miRNA species. RESULTS: The significance of miRNA-profiles was barely influenced by FFPE treatment in both tissues and the variance induced by FFPE treatment was much smaller than the variance caused by biologically based differential expression. CONCLUSION: FFPE material is well suited for miRNA profiling.