The PAX5-JAK2 translocation acts as dual-hit mutation that promotes aggressive B-cell leukemia via nuclear STAT5 activation.

While PAX5 is an important tumor suppressor gene in B-cell acute lymphoblastic leukemia (B-ALL), it is also involved in oncogenic translocations coding for diverse PAX5 fusion proteins. PAX5-JAK2 encodes a protein consisting of the PAX5 DNA-binding region fused to the constitutively active JAK2 kinase domain. Here, we studied the oncogenic function of the PAX5-JAK2 fusion protein in a mouse model expressing it from the endogenous Pax5 locus, resulting in inactivation of one of the two Pax5 alleles. Pax5Jak2/+ mice rapidly developed an aggressive B-ALL in the absence of another cooperating exogenous gene mutation. The DNA-binding function and kinase activity of Pax5-Jak2 as well as IL-7 signaling contributed to leukemia development. Interestingly, all Pax5Jak2/+ tumors lost the remaining wild-type Pax5 allele, allowing efficient DNA-binding of Pax5-Jak2. While we could not find evidence for a nuclear role of Pax5-Jak2 as an epigenetic regulator, high levels of active phosphorylated STAT5 and increased expression of STAT5 target genes were seen in Pax5Jak2/+ B-ALL tumors, implying that nuclear Pax5-Jak2 phosphorylates STAT5. Together, these data reveal Pax5-Jak2 as an important nuclear driver of leukemogenesis by maintaining phosphorylated STAT5 levels in the nucleus.

Pax5 regulates B cell immunity by promoting PI3K signaling via PTEN down-regulation.

The transcription factor Pax5 controls B cell development, but its role in mature B cells is largely enigmatic. Here, we demonstrated that the loss of Pax5 by conditional mutagenesis in peripheral B lymphocytes led to the strong reduction of B-1a, marginal zone (MZ), and germinal center (GC) B cells as well as plasma cells. Follicular (FO) B cells tolerated the loss of Pax5 but had a shortened half-life. The Pax5-deficient FO B cells failed to proliferate upon B cell receptor or Toll-like receptor stimulation due to impaired PI3K-AKT signaling, which was caused by increased expression of PTEN, a negative regulator of the PI3K pathway. Pax5 restrained PTEN protein expression at the posttranscriptional level, likely involving Pten-targeting microRNAs. Additional PTEN loss in Pten,Pax5 double-mutant mice rescued FO B cell numbers and the development of MZ B cells but did not restore GC B cell formation. Hence, the posttranscriptional down-regulation of PTEN expression is an important function of Pax5 that facilitates the differentiation and survival of mature B cells, thereby promoting humoral immunity.

The Immune Phenotype of Isolated Lymphoid Structures in Non-Tumorous Colon Mucosa Encrypts the Information on Pathobiology of Metastatic Colorectal Cancer.

The gut-associated lymphoid tissue represents an integral part of the immune system. Among the powerful players of the mucosa-associated lymphoid tissue are isolated lymphoid structures (ILSs), which as information centers, drive the local (and systemic) adaptive immune responses. Germinal center reactions, taking place within ILSs, involve the coordinated action of various immune cell types with a central role given to B cells. In the current study, we aimed at dissecting the impact of ILSs within non-tumorous colon tissue (NT) on the pathobiology of colorectal cancer (CRC) with metastasis in the liver (CRCLM). In particular, we focused on the immune phenotypes of ILSs and ectopic lymphoid structures (ELSs), built up at matching primary and metastatic tumor sites. We implemented an integrative analysis strategy on the basis of tissue image cytometry and clonality assessment to explore the immune phenotype of ILS/ELS at three tissue entities: NT, CRC, and CRCLM (69 specimens in total). Applying a panel of lineage markers used for immunostaining, we characterized and compared the anatomical features, the cellular composition, the activation, and proliferation status of ILSs and ELSs, and assessed the clinical relevance of staining-derived data sets. Our major discovery was that ILS characteristics at the NT site predefine the immune phenotype of ELSs at CRC and CRCLM. Thereby, B-cell-enriched (CD20) and highly proliferative (Ki67) ILSs and ELSs were found to be associated with improved clinical outcome in terms of survival and enabled patient stratification into risk groups. Moreover, the data revealed a linkage between B-cell clonality at the NT site and the metastatic characteristics of the tumor in the distant liver tissue. Consolidation of immunostaining-based findings with the results of compendium-wide transcriptomic analysis furthermore proposed CD27 as a novel marker of T follicular helper cells within lymphoid structures. Overall, the study nominates the ILS immune phenotype as a novel prognostic marker for patients with metastatic CRC.

Interrelations of Sphingolipid and Lysophosphatidate Signaling with Immune System in Ovarian Cancer.

The sphingolipid and lysophosphatidate regulatory networks impact diverse mechanisms attributed to cancer cells and the tumor immune microenvironment. Deciphering the complexity demands implementation of a holistic approach combined with higher-resolution techniques. We implemented a multi-modular integrative approach consolidating the latest accomplishments in gene expression profiling, prognostic/predictive modeling, next generation digital pathology, and systems biology for epithelial ovarian cancer. We assessed patient-specific transcriptional profiles using the sphingolipid/lysophosphatidate/immune-associated signature. This revealed novel sphingolipid/lysophosphatidate-immune gene-gene associations and distinguished tumor subtypes with immune high/low context. These were characterized by robust differences in sphingolipid-/lysophosphatidate-related checkpoints and the drug response. The analysis also nominates novel survival models for stratification of patients with CD68, LPAR3, SMPD1, PPAP2B, and SMPD2 emerging as the most prognostically important genes. Alignment of proprietary data with curated transcriptomic data from public databases across a variety of malignancies (over 600 categories; over 21,000 arrays) showed specificity for ovarian carcinoma. Our systems approach identified novel sphingolipid-lysophosphatidate-immune checkpoints and networks underlying tumor immune heterogeneity and disease outcomes. This holds great promise for delivering novel stratifying and targeting strategies.

Essential role for the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells.

Innate-like B-1a cells provide a first line of defense against pathogens, yet little is known about their transcriptional control. Here we identified an essential role for the transcription factor Bhlhe41, with a lesser contribution by Bhlhe40, in controlling B-1a cell differentiation. Bhlhe41-/-Bhlhe40-/- B-1a cells were present at much lower abundance than were their wild-type counterparts. Mutant B-1a cells exhibited an abnormal cell-surface phenotype and altered B cell receptor (BCR) repertoire exemplified by loss of the phosphatidylcholine-specific VH12Vκ4 BCR. Expression of a pre-rearranged VH12Vκ4 BCR failed to 'rescue' the mutant phenotype and revealed enhanced proliferation accompanied by increased cell death. Bhlhe41 directly repressed the expression of cell-cycle regulators and inhibitors of BCR signaling while enabling pro-survival cytokine signaling. Thus, Bhlhe41 controls the development, BCR repertoire and self-renewal of B-1a cells.

Molecular role of the PAX5-ETV6 oncoprotein in promoting B-cell acute lymphoblastic leukemia.

PAX5 is a tumor suppressor in B-ALL, while the role of PAX5 fusion proteins in B-ALL development is largely unknown. Here, we studied the function of PAX5-ETV6 and PAX5-FOXP1 in mice expressing these proteins from the Pax5 locus. Both proteins arrested B-lymphopoiesis at the pro-B to pre-B-cell transition and, contrary to their proposed dominant-negative role, did not interfere with the expression of most regulated Pax5 target genes. Pax5-Etv6, but not Pax5-Foxp1, cooperated with loss of the Cdkna2a/b tumor suppressors in promoting B-ALL development. Regulated Pax5-Etv6 target genes identified in these B-ALLs encode proteins implicated in pre-B-cell receptor (BCR) signaling and migration/adhesion, which could contribute to the proliferation, survival, and tissue infiltration of leukemic B cells. Together with similar observations made in human PAX5-ETV6+ B-ALLs, these data identified PAX5-ETV6 as a potent oncoprotein that drives B-cell leukemia development.

AID/APOBEC-network reconstruction identifies pathways associated with survival in ovarian cancer.

BACKGROUND: Building up of pathway-/disease-relevant signatures provides a persuasive tool for understanding the functional relevance of gene alterations and gene network associations in multifactorial human diseases. Ovarian cancer is a highly complex heterogeneous malignancy in respect of tumor anatomy, tumor microenvironment including pro-/antitumor immunity and inflammation; still, it is generally treated as single disease. Thus, further approaches to investigate novel aspects of ovarian cancer pathogenesis aiming to provide a personalized strategy to clinical decision making are of high priority. Herein we assessed the contribution of the AID/APOBEC family and their associated genes given the remarkable ability of AID and APOBECs to edit DNA/RNA, and as such, providing tools for genetic and epigenetic alterations potentially leading to reprogramming of tumor cells, stroma and immune cells. RESULTS: We structured the study by three consecutive analytical modules, which include the multigene-based expression profiling in a cohort of patients with primary serous ovarian cancer using a self-created AID/APOBEC-associated gene signature, building up of multivariable survival models with high predictive accuracy and nomination of top-ranked candidate/target genes according to their prognostic impact, and systems biology-based reconstruction of the AID/APOBEC-driven disease-relevant mechanisms using transcriptomics data from ovarian cancer samples. We demonstrated that inclusion of the AID/APOBEC signature-based variables significantly improves the clinicopathological variables-based survival prognostication allowing significant patient stratification. Furthermore, several of the profiling-derived variables such as ID3, PTPRC/CD45, AID, APOBEC3G, and ID2 exceed the prognostic impact of some clinicopathological variables. We next extended the signature-/modeling-based knowledge by extracting top genes co-regulated with target molecules in ovarian cancer tissues and dissected potential networks/pathways/regulators contributing to pathomechanisms. We thereby revealed that the AID/APOBEC-related network in ovarian cancer is particularly associated with remodeling/fibrotic pathways, altered immune response, and autoimmune disorders with inflammatory background. CONCLUSIONS: The herein study is, to our knowledge, the first one linking expression of entire AID/APOBECs and interacting genes with clinical outcome with respect to survival of cancer patients. Overall, data propose a novel AID/APOBEC-derived survival model for patient risk assessment and reconstitute mapping to molecular pathways. The established study algorithm can be applied further for any biologically relevant signature and any type of diseased tissue.

Exploring the role of sphingolipid machinery during the epithelial to mesenchymal transition program using an integrative approach.

The epithelial to mesenchymal transition (EMT) program is activated in epithelial cancer cells and facilitates their ability to metastasize based on enhanced migratory, proliferative, anti-apoptotic, and pluripotent capacities. Given the fundamental impact of sphingolipid machinery to each individual process, the sphingolipid-related mechanisms might be considered among the most prominent drivers/players of EMT; yet, there is still limited knowledge. Given the complexity of the interconnected sphingolipid system, which includes distinct sphingolipid mediators, their synthesizing enzymes, receptors and transporters, we herein apply an integrative approach for assessment of the sphingolipid-associated mechanisms underlying EMT program. We created the sphingolipid-/EMT-relevant 41-gene/23-gene signatures which were applied to denote transcriptional events in a lung cancer cell-based EMT model. Based on defined 35-gene sphingolipid/EMT-attributed signature of regulated genes, we show close associations between EMT markers, genes comprising the sphingolipid network at multiple levels and encoding sphingosine 1-phosphate (S1P)-/ceramide-metabolizing enzymes, S1P and lysophosphatidic acid (LPA) receptors and S1P transporters, pluripotency genes and inflammation-related molecules, and demonstrate the underlying biological pathways and regulators. Mass spectrometry-based sphingolipid analysis revealed an EMT-attributed shift towards increased S1P and LPA accompanied by reduced ceramide levels. Notably, using transcriptomics data across various cell-based perturbations and neoplastic tissues (24193 arrays), we identified the sphingolipid/EMT signature primarily in lung adenocarcinoma tissues; besides, bladder, colorectal and prostate cancers were among the top-ranked. The findings also highlight novel regulatory associations between influenza virus and the sphingolipid/EMT-associated mechanisms. In sum, data propose the multidimensional contribution of sphingolipid machinery to pathological EMT and may yield new biomarkers and therapeutic targets.

Wapl is an essential regulator of chromatin structure and chromosome segregation.

Mammalian genomes contain several billion base pairs of DNA that are packaged in chromatin fibres. At selected gene loci, cohesin complexes have been proposed to arrange these fibres into higher-order structures, but how important this function is for determining overall chromosome architecture and how the process is regulated are not well understood. Using conditional mutagenesis in the mouse, here we show that depletion of the cohesin-associated protein Wapl stably locks cohesin on DNA, leads to clustering of cohesin in axial structures, and causes chromatin condensation in interphase chromosomes. These findings reveal that the stability of cohesin-DNA interactions is an important determinant of chromatin structure, and indicate that cohesin has an architectural role in interphase chromosome territories. Furthermore, we show that regulation of cohesin-DNA interactions by Wapl is important for embryonic development, expression of genes such as c-myc (also known as Myc), and cell cycle progression. In mitosis, Wapl-mediated release of cohesin from DNA is essential for proper chromosome segregation and protects cohesin from cleavage by the protease separase, thus enabling mitotic exit in the presence of functional cohesin complexes.

An RNA-Seq strategy to detect the complete coding and non-coding transcriptome including full-length imprinted macro ncRNAs.

Imprinted macro non-protein-coding (nc) RNAs are cis-repressor transcripts that silence multiple genes in at least three imprinted gene clusters in the mouse genome. Similar macro or long ncRNAs are abundant in the mammalian genome. Here we present the full coding and non-coding transcriptome of two mouse tissues: differentiated ES cells and fetal head using an optimized RNA-Seq strategy. The data produced is highly reproducible in different sequencing locations and is able to detect the full length of imprinted macro ncRNAs such as Airn and Kcnq1ot1, whose length ranges between 80-118 kb. Transcripts show a more uniform read coverage when RNA is fragmented with RNA hydrolysis compared with cDNA fragmentation by shearing. Irrespective of the fragmentation method, all coding and non-coding transcripts longer than 8 kb show a gradual loss of sequencing tags towards the 3' end. Comparisons to published RNA-Seq datasets show that the strategy presented here is more efficient in detecting known functional imprinted macro ncRNAs and also indicate that standardization of RNA preparation protocols would increase the comparability of the transcriptome between different RNA-Seq datasets.

Activation-induced cytidine deaminase (AID)-associated multigene signature to assess impact of AID in etiology of diseases with inflammatory component.

Activation-induced cytidine deaminase (AID) is expressed in B cells within germinal centers and is critically involved in class switch recombination and somatic hypermutation of immunoglobulin loci. Functionally active AID can additionally be detected within ectopic follicular structures developed at sites of chronic inflammation. Furthermore, AID may target non-Ig genes in B- and non-B-cell background. Therefore, AID-associated effects are of increasing interest in disease areas such as allergy, inflammation, autoimmunity, and cancer.Pathway- or disease-relevant multigene signatures have attracted substantial attention for therapeutic target proposal, diagnostic tools, and monitoring of therapy response. To delineate the impact of AID in etiology of multifactorial diseases, we designed the AID-associated 25-gene signature. Chronic rhinosinusitis with nasal polyps was used as an inflammation-driven airway disease model; high levels of IgE have been previously shown to be present within polyp tissue. Expression levels of 16 genes were found to be modulated in polyps including AID, IgG and IgE mature transcripts which reflect AID activity; clustering algorithm revealed an AID-specific gene signature for the disease state with nasal polyp. Complementary, AID-positive ectopic lymphoid structures were detected within polyp tissues by in situ immunostaining. Our data demonstrate the class switch recombination and somatic hypermutation events likely taking place locally in the airways and in addition to the previously highlighted markers and/or targets as IL5 and IgE suggest novel candidate genes to be considered for treatment of nasal polyposis including among others IL13 and CD23. Thus, the algorithm presented herein including the multigene signature approach, analysis of co-regularities and creation of AID-associated functional network gives an integrated view of biological processes and might be further applied to assess role of altered AID expression in etiology of other diseases, in particular, aberrant immunity and cancer.

The transcription factor Pax5 regulates its target genes by recruiting chromatin-modifying proteins in committed B cells.

Pax5 is a critical regulator of B-cell commitment. Here, we identified direct Pax5 target genes by streptavidin-mediated ChIP-chip analysis of pro-B cells expressing in vivo biotinylated Pax5. By binding to promoters and enhancers, Pax5 directly regulates the expression of multiple transcription factor, cell surface receptor and signal transducer genes. One of the newly identified enhancers was shown by transgenic analysis to confer Pax5-dependent B-cell-specific activity to the Nedd9 gene controlling B-cell trafficking. Profiling of histone modifications in Pax5-deficient and wild-type pro-B cells demonstrated that Pax5 induces active chromatin at activated target genes, while eliminating active chromatin at repressed genes in committed pro-B cells. Pax5 rapidly induces these chromatin and transcription changes by recruiting chromatin-remodelling, histone-modifying and basal transcription factor complexes to its target genes. These data provide novel insight into the regulatory network and epigenetic regulation, by which Pax5 controls B-cell commitment.

Transcriptional repression of ceramide kinase in LPS-challenged macrophages.

Ceramide kinase (CERK) produces the bioactive lipid ceramide 1-phosphate (C1P). Both CERK and C1P have been identified as mediators of cell growth and survival. Recent evidence showed that CERK is down-regulated during M1-type macrophage activation, which is known to promote cell growth arrest. However, the mechanism has not been investigated yet and, in particular, whether growth arrest might be the signal for down-regulation of CERK is currently unknown. Here, we found that LPS-mediated TLR-4 engagement reduces Cerk mRNA levels in mouse primary macrophages. Reporter gene assays in RAW264.7 macrophages showed that LPS inhibits the transcriptional activity of the Cerk proximal promoter. The G1-cell cycle blocker mimosine did not inhibit Cerk transcription, suggesting that transcriptional repression of Cerk by LPS is not a primary consequence of LPS-induced cell cycle blockade.

Polycomb complexes act redundantly to repress genomic repeats and genes.

Polycomb complexes establish chromatin modifications for maintaining gene repression and are essential for embryonic development in mice. Here we use pluripotent embryonic stem (ES) cells to demonstrate an unexpected redundancy between Polycomb-repressive complex 1 (PRC1) and PRC2 during the formation of differentiated cells. ES cells lacking the function of either PRC1 or PRC2 can differentiate into cells of the three germ layers, whereas simultaneous loss of PRC1 and PRC2 abrogates differentiation. On the molecular level, the differentiation defect is caused by the derepression of a set of genes that is redundantly repressed by PRC1 and PRC2 in ES cells. Furthermore, we find that genomic repeats are Polycomb targets and show that, in the absence of Polycomb complexes, endogenous murine leukemia virus elements can mobilize. This indicates a contribution of the Polycomb group system to the defense against parasitic DNA, and a potential role of genomic repeats in Polycomb-mediated gene regulation.

Subcellular localization of ceramide kinase and ceramide kinase-like protein requires interplay of their Pleckstrin Homology domain-containing N-terminal regions together with C-terminal domains.

Ceramide kinase (CERK) and the ceramide kinase-like protein (CERKL), two related members of the diacylglycerol kinase family, are ill-defined at the molecular level. In particular, what determines their distinctive subcellular localization is not well understood. Here we show that the Pleckstrin Homology (PH) domain of CERK, which is required for Golgi complex localization, can substitute for the N-terminal region of CERKL and allow for wild-type CERKL localization, which is typified by nucleolar accumulation. This demonstrates that determinants for localization of these two enzymes do not lie solely in their PH domain-containing N-terminal regions. Moreover, we present evidence for a previously unrecognized participation of CERK distal sequences in structural stability, localization and activity of the full-length protein. Progressive deletion of CERK and CERKL from the C-terminus revealed similar sequential organization in both proteins, with nuclear import signals in their N-terminal part, and nuclear export signals in their C-terminal part. Furthermore, mutagenesis of individual cysteine residues of a CERK-specific CXXXCXXC motif severely compromised both exportation of CERK from the nucleus and its association with the Golgi complex. Altogether, this work identifies conserved domains in CERK and CERKL as well as new determinants for their subcellular localization. It further suggests a nucleocytoplasmic shuttling mechanism for both proteins that may be defective in CERKL mutant proteins responsible for retinal degenerative diseases.

A conserved cysteine motif essential for ceramide kinase function.

Ceramide kinase (CerK) is a sphingolipid metabolizing enzyme very sensitive to oxidation; however, the determinants are unknown. We show here that the thiol-modifying agent N-ethyl-maleimide abrogates CerK activity in vitro and in a cell based assay, implying that important cysteine residues are accessible in purified as well as endogenous CerK. We replaced every 22 residues in human CerK, by an alanine, and measured activity in the resulting mutant proteins. This led to identification of a cluster of cysteines, C(347)XXXC(351)XXC(354), essential for CerK function. These findings are discussed based on homology modeling of the catalytic domain of CerK.

Assessing the function of human UNC-93B in Toll-like receptor signaling and major histocompatibility complex II response.

The high sequence identity observed between UNC-93B of mouse and human imply common evolutionary ancestors and a conserved function. A nonconservative point mutation in the mouse Unc93b1 gene has been associated with defective Toll-like receptor (TLR) signaling and impaired major histocompatibility complex (MHC) I and II restricted antigen responses. Like murine UNC-93B, the human homologue is predicted to form 12 transmembrane domains, and it localizes to the endoplasmic reticulum. In human beings its expression is highest in professional antigen-presenting cells such as dendritic cells and macrophages. Interestingly, UNC-93B itself is specifically induced by TLR3 signaling in monocyte-derived dendritic cells and macrophages. To study the effect of UNC-93B deficiency in TLR signaling and antigen-presentation in human beings, UNC-93B message was knocked down in monocyte-derived dendritic cells and a reduced TNFalpha production in response to TLR3 agonists was observed. In the same experiment, the achieved knockdown had no effect on an MHC II-dependent antigen response, suggesting that the reduced quantity of human UNC-93B was still capable of supporting class II antigen presentation or that UNC-93B is not required for class II antigen presentation in human antigen-presenting cells.

A critical beta6-beta7 loop in the pleckstrin homology domain of ceramide kinase.

CerK (ceramide kinase) produces ceramide 1-phosphate, a sphingophospholipid with recognized signalling properties. It localizes to the Golgi complex and fractionates essentially between detergent-soluble and -insoluble fractions; however, the determinants are unknown. Here, we made a detailed mutagenesis study of the N-terminal PH domain (pleckstrin homology domain) of CerK, based on modelling, and identified key positively charged amino acid residues within an unusual motif in the loop interconnecting beta-strands 6 and 7. These residues are critical for CerK membrane association and polyphosphoinositide binding and activity. Their mutagenesis results in increased thermolability, sensitivity to proteolysis, reduced apparent molecular mass as well as propensity of the recombinant mutant protein to aggregate, indicating that this loop impacts the overall conformation of the CerK protein. This is in contrast with most PH domains whose function strongly relies on charges located in the beta1-beta2 loop.

mRNA openers and closers: modulating AU-rich element-controlled mRNA stability by a molecular switch in mRNA secondary structure.

Approximately 3 000 genes are regulated in a time-, tissue-, and stimulus-dependent manner by degradation or stabilization of their mRNAs. The process is mediated by interaction of AU-rich elements (AREs) in the mRNA's 3'-untranslated regions with trans-acting factors. AU-rich element-controlled genes of fundamentally different functional relevance depend for their activation on one positive regulator, HuR. Here we present a methodology to exploit this central regulatory process for specific manipulation of AU-rich element-controlled gene expression at the mRNA level. With a combination of single-molecule spectroscopy, computational biology, and molecular and cellular biochemistry, we show that mRNA recognition by HuR is dependent on the presentation of the sequence motif NNUUNNUUU in single-stranded conformation. The presentation of the HuR binding site in the mRNA secondary structure appears to act analogously to a regulatory on/off switch that specifically controls HuR access to mRNAs in cis. Based on this finding we present a methodology for manipulating ARE mRNA levels by actuating this conformational switch specifically in a target mRNA. Computationally designed oligonucleotides (openers) enhance the NNUUNNUUU accessibility by rearranging the mRNA conformation. Thereby they increase in vitro and endogenous HuR-mRNA complex formation which leads to specific mRNA stabilization (as demonstrated for TNFalpha and IL-2, respectively). Induced HuR binding both inside and outside the AU-rich element promotes functional IL-2 mRNA stabilization. This opener-induced mRNA stabilization mimics the endogenous IL-2 response to CD28 stimulation in human primary T-cells. We therefore propose that controlled modulation of the AU-rich element conformation by mRNA openers or closers allows message stabilization or destabilization in cis to be specifically triggered. The described methodology might provide a means for studying distinct pathways in a complex cellular network at the node of mRNA stability control. It allows ARE gene expression to be potentially silenced or boosted. This will be of particular value for drug-target validation, allowing the diseased phenotype to ameliorate or deteriorate. Finally, the mRNA openers provide a rational starting point for target-specific mRNA stability assays to screen for low-molecular-weight compounds acting as inhibitors or activators of an mRNA structure rearrangement.