Inducing mRNA Decay Research Articles

The oncogenic role of EIF4A3/CDC20 axis in the endometrial cancer.

Eukaryotic initiation factor 4A-3 (EIF4A3) is a key component of the exon junction complex (EJC) and is extensively involved in RNA splicing, inducing mRNA decay, and regulating the cell cycle and apoptosis. However, the potential role of EIF4A3 in EC has not been comprehensively investigated and remains unknown. Here, we report that the expression level of EIF4A3 is dramatically elevated in endometrial cancer (EC) samples compared with normal EC samples via bioinformatics analysis and immunohistochemistry analysis, and that high expression of EIF4A3 promotes the proliferation, migration, and invasion of EC cells. Mechanistically, we found that high EIF4A3 expression stabilized cell division cyclin 20 (CDC20) mRNA, and high EIF4A3 expression induced pro-carcinogenic effects in EC cells that were efficiently antagonized upon knockdown of CDC20, as well as Apcin, an inhibitor of CDC20. These findings reveal a novel mechanism by which high expression of EIF4A3 induces CDC20 upregulation, thus leading to EC tumorigenesis and metastasis, indicating a potential treatment strategy for EC patients with high EIF4A3 expression using Apcin. KEY MESSAGES: The expression level of EIF4A3 was dramatically elevated in endometrial cancer (EC) samples compared with normal endometrial cancer samples. High EIF4A3 expression stabilized CDC20 mRNA, and high EIF4A3 expression induced pro-carcinogenic effect in EC cells which was efficiently antagonized upon knockdown of CDC20. Apcin, an inhibitor of CDC20, could effectively counteract high expression of EIF4A3 inducing EC tumourigenesis and metastasis, indicating the potential treatment strategy for EC patients with EIF4A3 high expression by using Apcin.

Engineered Protease-Responsive RNA-Binding Proteins (RBPs) to Expand the Toolbox of Synthetic Circuits in Mammalian Cells.

Genetically encoded sensor-actuator circuits aim at reprogramming cellular functions and are inspired by intracellular networks: from the input signal (sensor) to the desired output response (actuator). In the last years, circuits with posttranscriptional regulation of gene expression have aroused great interest for their potential in the biomedical space. Posttranscriptional modulation can be achieved with ribozymes, riboswitches (simple regulatory elements based on RNA secondary structures), noncoding RNAs, and RNA-binding proteins (RBPs). RBPs are proteins that recognize specific motifs on the mRNA target inducing mRNA decay or translation inhibition. The use of RBPs deriving from different species in mammalian cells has allowed to create sophisticated and multilayered regulatory networks, addressing the previous limitation of regulatory orthogonal parts that can be assembled in synthetic devices. In this chapter, we describe the engineering and tests of protease-responsive RNA-binding proteins (L7Ae and MS2-cNOT7) to expand the toolbox of synthetic circuits in mammalian cells.

Prenatal alcohol exposure disrupts miR‐150‐5p regulation of angiogenesis and blood‐brain barrier integrity genes

Fetal alcohol spectrum disorders (FASD) occur after in utero ethanol (EtOH) exposure and manifest in a wide range of neurocognitive deficits. These deficits can be caused by EtOH-induced dysregulation of genes required for angiogenesis and vascular integrity which are being regulated by microRNAs (miRNAs). miRNAs silence gene expression by binding to the 3’UTRs of target mRNAs, inducing mRNA decay. In an established mouse model of moderate prenatal alcohol exposure (PAE), we found that cortices (CTX) and primary brain microvascular endothelial cells (pBMVECs) isolated from PAE pups at embryonic day 18 (E18) had significantly higher levels of miR-150-5p compared to saccharin-exposed controls. A novel angiogenic target of miR-150-5p, vascular endothelial zinc finger 1 (Vezf1), is an endothelial-specific transcription factor required for vascular development. Vezf1 levels were significantly reduced in E18 PAE CTX and pBMVECs compared to controls. pBMVECs also showed a decrease in expression of other genes important for vascular function and integrity, including Claudin-12, Claudin-5, Occludin, VE-cadherin and MMP14. To identify the effects of EtOH on endothelial cell gene expression, we treated a mouse BMVE cell line (BMVECs) with 25mM of EtOH for 24 hrs. miR-150-5p expression increased after EtOH treatment compared to no treatment controls. Transfection of miR-150-5p LNATM mimics in BMVECs decreased Vezf1, Claudin-12 and MMP14 expression, while miR-150-5p LNATM inhibitors increased their expression. To demonstrate direct interaction between miR-150-5p and Vezf1, we cloned the Vezf1 3’UTR into a firefly luciferase reporter. Dual luciferase assays showed reduced luciferase activity for the Vezf1 3’UTR reporter with miR-150-5p LNATM mimics. To investigate the effect of EtOH and miR-150-5p on endothelial cell function, we performed in vitro migration, tube formation and permeability assays. BMVECs treated with EtOH significantly attenuated migration and tube formation vs no EtOH treatment controls. In addition, miR-150-5p overexpression in BMVECs attenuated tube formation and cell migration, and significantly increased cell permeability. Correspondingly, miR-150-5p inhibition significantly enhanced tube formation and migration, and decreased cell permeability. Our research suggests that miR-150-5p may adversely alter the cortical microvasculature during PAE by inhibiting the expression of targets important for angiogenesis and blood-brain barrier integrity.

MicroRNA Transcriptome Profiling in Heart of Trypanosoma cruzi-Infected Mice.

MicroRNAs (miRNAs) are a class of small noncoding RNAs (typically 19-23 nucleotides) which act by annealing to partially complementary binding sites present on the 3' untranslated regions (UTR) of messenger RNAs (mRNAs) leading to inhibition of protein translation or by inducing mRNA decay. Since their discovery, miRNAs have come to be recognized as master regulators of gene expression in plant and mammals, controlling tissue-specific protein expression. Up to one-third of mammalian mRNAs are susceptible to miRNA-mediated regulation. It has been shown that miRNAs are determinants of the physiology and pathophysiology of the cardiovascular system, and altered expression of muscle- and/or cardiac-specific miRNAs in myocardial tissue is involved in heart development and cardiovascular diseases, including myocardial hypertrophy, heart failure, and fibrosis. The analysis of miRNA expression pattern provides important information, as well as is a starting point to understand miRNA function in different tissues, during development, and in disease. Several techniques can be used for miRNA profiling analysis like high-throughput sequencing, microarrays, and real-time PCR using microfluidic low-density arrays. This chapter describes the complete methodology to perform miRNA profiling using the stem-loop reverse-transcription (RT)-based TaqMan® MicroRNA low-density arrays (TLDA) method. This methodology was used to perform miRNA profiling in the heart of T. cruzi acutely infected mice.

Arc 3' UTR Splicing Leads to Dual and Antagonistic Effects in Fine-Tuning Arc Expression Upon BDNF Signaling.

Activity-regulated cytoskeletal associated protein (Arc) is an immediate-early gene critically involved in synaptic plasticity and memory consolidation. Arc mRNA is rapidly induced by synaptic activation and a portion is locally translated in dendrites where it modulates synaptic strength. Being an activity-dependent effector of homeostatic balance, regulation of Arc is uniquely tuned to result in short-lived bursts of expression. Cis-Acting elements that control its transitory expression post-transcriptionally reside primarily in Arc mRNA 3′ UTR. These include two conserved introns which distinctively modulate Arc mRNA stability by targeting it for destruction via the nonsense mediated decay pathway. Here, we further investigated how splicing of the Arc mRNA 3′ UTR region contributes to modulate Arc expression in cultured neurons. Unexpectedly, upon induction with brain derived neurotrophic factor, translational efficiency of a luciferase reporter construct harboring Arc 3′ UTR is significantly upregulated and this effect is dependent on splicing of Arc introns. We find that, eIF2α dephosphorylation, mTOR, ERK, PKC, and PKA activity are key to this process. Additionally, CREB-dependent transcription is required to couple Arc 3′ UTR-splicing to its translational upregulation, suggesting the involvement of de novo transcribed trans-acting factors. Overall, splicing of Arc 3′ UTR exerts a dual and unique effect in fine-tuning Arc expression upon synaptic signaling: while inducing mRNA decay to limit the time window of Arc expression, it also elicits translation of the decaying mRNA. This antagonistic effect likely contributes to the achievement of a confined yet efficient burst of Arc protein expression, facilitating its role as an effector of synapse-specific plasticity.

Binding of NUFIP2 to Roquin promotes recognition and regulation of ICOS mRNA

The ubiquitously expressed RNA-binding proteins Roquin-1 and Roquin-2 are essential for appropriate immune cell function and postnatal survival of mice. Roquin proteins repress target mRNAs by recognizing secondary structures in their 3′-UTRs and by inducing mRNA decay. However, it is unknown if other cellular proteins contribute to target control. To identify cofactors of Roquin, we used RNA interference to screen ~1500 genes involved in RNA-binding or mRNA degradation, and identified NUFIP2 as a cofactor of Roquin-induced mRNA decay. NUFIP2 binds directly and with high affinity to Roquin, which stabilizes NUFIP2 in cells. Post-transcriptional repression of human ICOS by endogenous Roquin proteins requires two neighboring non-canonical stem-loops in the ICOS 3′-UTR. This unconventional cis-element as well as another tandem loop known to confer Roquin-mediated regulation of the Ox40 3′-UTR, are bound cooperatively by Roquin and NUFIP2. NUFIP2 therefore emerges as a cofactor that contributes to mRNA target recognition by Roquin.

Soaking up tumor suppression

Cancer Biology MicroRNAs (miRNAs) regulate gene expression by inducing mRNA decay. But miRNAs can be regulated through other RNAs and DNA that bind and sequester them without inducing any mRNA decay; these are known as miRNA sponges. Gilot et al. show that tyrosinase-related protein 1 (TYRP1) mRNA

MiR-222 in Cardiovascular Diseases: Physiology and Pathology.

MicroRNAs (miRNAs and miRs) are endogenous 19–22 nucleotide, small noncoding RNAs with highly conservative and tissue specific expression. They can negatively modulate target gene expressions through decreasing transcription or posttranscriptional inducing mRNA decay. Increasing evidence suggests that deregulated miRNAs play an important role in the genesis of cardiovascular diseases. Additionally, circulating miRNAs can be biomarkers for cardiovascular diseases. MiR-222 has been reported to play important roles in a variety of physiological and pathological processes in the heart. Here we reviewed the recent studies about the roles of miR-222 in cardiovascular diseases. MiR-222 may be a potential cardiovascular biomarker and a new therapeutic target in cardiovascular diseases.

Activating protein phosphatase 2A (PP2A) enhances tristetraprolin (TTP) anti-inflammatory function in A549 lung epithelial cells

Chronic respiratory diseases are driven by inflammation, but some clinical conditions (severe asthma, COPD) are refractory to conventional anti-inflammatory therapies. Thus, novel anti-inflammatory strategies are necessary. The mRNA destabilizing protein, tristetraprolin (TTP), is an anti-inflammatory molecule that functions to induce mRNA decay of cytokines that drive pathogenesis of respiratory disorders. TTP is regulated by phosphorylation and protein phosphatase 2A (PP2A) is responsible for dephosphorylating (and hence activating) TTP, amongst other targets. PP2A is activated by small molecules, FTY720 and AAL(S), and in this study we examine whether these compounds repress cytokine production in a cellular model of airway inflammation using A549 lung epithelial cells stimulated with tumor necrosis factor α (TNFα) in vitro. PP2A activators significantly increase TNFα-induced PP2A activity and inhibit mRNA expression and protein secretion of interleukin 8 (IL-8) and IL-6; two key pro-inflammatory cytokines implicated in respiratory disease and TTP targets. The effect of PP2A activators is not via an increase in TNFα-induced TTP mRNA expression; instead we demonstrate a link between PP2A activation and TTP anti-inflammatory function by showing that specific knockdown of TTP with siRNA reversed the repression of TNFα-induced IL-8 and IL-6 mRNA expression and protein secretion by FTY720. Therefore we propose that PP2A activators affect the dynamic equilibrium regulating TTP; shifting the equilibrium from phosphorylated (inactive) towards unphosphorylated (active) but unstable TTP. PP2A activators boost the anti-inflammatory function of TTP and have implications for future pharmacotherapeutic strategies to combat inflammation in respiratory disease.

Emerging Roles of CCCH-Type Zinc Finger Proteins in Destabilizing mRNA Encoding Inflammatory Factors and Regulating Immune Responses.

Posttranscriptional gene regulation is a rapid and effective way to mediate the expression of inflammatory genes. CCCH-type zinc finger proteins are nucleotide-binding molecules involved in RNA metabolism pathways such as RNA splicing, polyadenylation, and messenger RNA (mRNA) decay. Among these proteins, tristetraproline, Roquins, and Regnase-1/monocyte chemotactic protein-1-induced protein-1 have been recently reported to be responsible for mRNA instability. They bind to mRNAs harboring unique motifs and induce mRNA decay. In this review we summarize current progress regarding the specific characteristics of sequences and structures in the 3' untranslated regions of mRNAs that are recognized by tristetraproline, Roquins, and Regnase-1. The target mRNAs to be destabilized by those CCCH-type zinc finger proteins also are included. Notably, most target mRNAs encode cytokines and other inflammatory mediators, suggesting the immune regulation role of CCCH zinc finger proteins. Mice carrying a genetic null allele or modification of these genes display severe symptoms of autoimmune diseases. Taken together, data show that CCCH-type zinc finger proteins play a crucial role in regulating immune response by targeting multiple mRNAs, and including decay. Further understanding the functions of these proteins may provide new therapeutic targets for immune-related disorders in the future.

A post-translational regulatory switch on UPF1 controls targeted mRNA degradation.

Nonsense-mediated mRNA decay (NMD) controls the quality of eukaryotic gene expression and also degrades physiologic mRNAs. How NMD targets are identified is incompletely understood. A central NMD factor is the ATP-dependent RNA helicase upframeshift 1 (UPF1). Neither the distance in space between the termination codon and the poly(A) tail nor the binding of steady-state, largely hypophosphorylated UPF1 is a discriminating marker of cellular NMD targets, unlike for premature termination codon (PTC)-containing reporter mRNAs when compared with their PTC-free counterparts. Here, we map phosphorylated UPF1 (p-UPF1)-binding sites using transcriptome-wide footprinting or DNA oligonucleotide-directed mRNA cleavage to report that p-UPF1 provides the first reliable cellular NMD target marker. p-UPF1 is enriched on NMD target 3' untranslated regions (UTRs) along with suppressor with morphogenic effect on genitalia 5 (SMG5) and SMG7 but not SMG1 or SMG6. Immunoprecipitations of UPF1 variants deficient in various aspects of the NMD process in parallel with Förster resonance energy transfer (FRET) experiments reveal that ATPase/helicase-deficient UPF1 manifests high levels of RNA binding and disregulated hyperphosphorylation, whereas wild-type UPF1 releases from nonspecific RNA interactions in an ATP hydrolysis-dependent mechanism until an NMD target is identified. 3' UTR-associated UPF1 undergoes regulated phosphorylation on NMD targets, providing a binding platform for mRNA degradative activities. p-UPF1 binding to NMD target 3' UTRs is stabilized by SMG5 and SMG7. Our results help to explain why steady-state UPF1 binding is not a marker for cellular NMD substrates and how this binding is transformed to induce mRNA decay.

Smaug destroys a huge treasure

Smaug, a protein repressing translation and inducing mRNA decay, directly controls an unexpectedly large number of maternal mRNAs driving early Drosophila development.See related research, http://genomebiology.com/2014/15/1/R4

Tumor Suppressor MiR-29b Targets HDAC4 and Modulates Vorinostat Activity In Multiple Myeloma Cells

MicroRNAs (miRNAs) are short non-coding RNAs that control gene expression at the post-transcriptional level by inducing mRNA decay or translation repression. A subclass of miRNAs, named epi-miRNAs, is known to exert anti-tumor activity by targeting effectors of the epigenetic machinery. We recently demonstrated a key role for the tumor suppressor miR-29b in reducing the global DNA methylation of multiple myeloma (MM) cells through the targeting of DNA-methyltransferases. In silico search of additional miR-29b targets contributing to clarify its role as epi-miRNA unveiled the class II histone deacetylase HDAC4. Since histone deacetylases represent promising molecular targets for cancer treatment, we sought to characterize HDAC4 expression and function and its regulation by miR-29b in MM cells. HDAC4 protein levels and enzymatic activity were found up-regulated in a panel of 11 MM cell lines as compared to peripheral blood mononuclear cells from healthy donors. Moreover, the analysis of HDAC4 mRNA levels in a microarray dataset consisting of 4 healthy controls, 55 MM and 29 plasma cell leukemia patients indicated significant over-expression in cancer samples, suggesting that high HDAC4 expression might be involved in MM pathogenesis. Notably, the analysis of miRNA/mRNA paired expression in the same microarray dataset revealed an inverse correlation between miR-29b and HDAC4 mRNA, strengthening the relevance of miR-29b as a key regulator of HDAC4. Synthetic miR-29b mimics transfected in MM cells (SKMM1 and NCI-H929) down-regulated HDAC4 mRNA and protein levels and inhibited the 3’UTR of HDAC4 cloned in a luciferase reporter vector, whereas failed to regulate a 3’UTR devoid of two predicted miR-29b target sites. On the other hand, lentiviral-mediated overexpression of HDAC4 strongly inhibited miR-29b expression. These results underscore a negative feedback loop occurring between miR-29b and its target HDAC4 in MM cells. Through loss of function experiments, we assessed the functional significance of HDAC4 in MM cells. Stable silencing of HDAC4 by shRNAs induced growth inhibition, caspase 3/7-dependent apoptosis and autophagy in U266 and KMS11 cells, which occurred together to miR-29b up-regulation. Interestingly, the pan-HDAC inhibitor vorinostat also triggered apoptosis and autophagy in MM cells, along with the induction of miR-29b and the down-regulation of HDAC4 and other miR-29b-canonical targets like CDK6, MCL-1 and Sp1. miR-29b itself was able to promote autophagy, as assessed by beclin-1 up-regulation and LC3A/B proteolytic cleavage in miR-29b mimics-transfected MM cells, which was abrogated by constitutive expression of HDAC4. Finally, we provided evidence that miR-29b over-expression potentiated, whereas its stable inhibition dampened, apoptosis and autophagy triggered by vorinostat. In conclusion, our findings shed light on the oncogenic role of HDAC4, which can be targeted through miR-29b-based therapeutic approaches, and identify miR-29b as a relevant effector of vorinostat activity in MM cells. Disclosures: No relevant conflicts of interest to declare.

Cytoplasmic deadenylation: regulation of mRNA fate

The poly(A) tail of mRNA has an important influence on the dynamics of gene expression. On one hand, it promotes enhanced mRNA stability to allow production of the protein, even after inactivation of transcription. On the other hand, shortening of the poly(A) tail (deadenylation) slows down translation of the mRNA, or prevents it entirely, by inducing mRNA decay. Thus deadenylation plays a crucial role in the post-transcriptional regulation of gene expression, deciding the fate of individual mRNAs. It acts both in basal mRNA turnover, as well as in temporally and spatially regulated translation and decay of specific mRNAs. In the present paper, we discuss mRNA deadenylation in eukaryotes, focusing on the main deadenylase, the Ccr4-Not complex, including its composition, regulation and functional roles.

Stress granules

Stress granules

Regulation of Epidermal Growth Factor Receptor Signaling in Human Cancer Cells by MicroRNA-7

The epidermal growth factor receptor (EGFR) is frequently overexpressed in cancer and is an important therapeutic target. Aberrant expression and function of microRNAs have been associated with tumorigenesis. Bioinformatic predictions suggest that the human EGFR mRNA 3'-untranslated region contains three microRNA-7 (miR-7) target sites, which are not conserved across mammals. We found that miR-7 down-regulates EGFR mRNA and protein expression in cancer cell lines (lung, breast, and glioblastoma) via two of the three sites, inducing cell cycle arrest and cell death. Because miR-7 was shown to decrease EGFR mRNA expression, we used microarray analysis to identify additional mRNA targets of miR-7. These included Raf1 and multiple other genes involved in EGFR signaling and tumorigenesis. Furthermore, miR-7 attenuated activation of protein kinase B (Akt) and extracellular signal-regulated kinase 1/2, two critical effectors of EGFR signaling, in different cancer cell lines. These data establish an important role for miR-7 in controlling mRNA expression and indicate that miR-7 has the ability to coordinately regulate EGFR signaling in multiple human cancer cell types.

Genome-wide Analysis Identifies Interleukin-10 mRNA as Target of Tristetraprolin

Tristetraprolin (TTP) is an RNA-binding protein required for the rapid degradation of mRNAs containing AU-rich elements. Targets regulated by TTP include the mRNAs encoding tumor necrosis factor-alpha, granulocyte-macrophage colony-stimulating factor, interleukin-2 (IL-2), and immediate early response 3. To identify novel target mRNAs of TTP in macrophages, we used a genome-wide approach that combines RNA immunoprecipitation and microarray analysis. A list was compiled of 137 mRNAs that are associated with TTP with an estimated accuracy on the order of 90%. Sequence analysis revealed a highly significant enrichment of AU-rich element motifs, with AUUUA pentamers present in 96% and UUAUUUAUU nonamers present in 44% of TTP-associated mRNAs. We further show that IL-10 is a novel target regulated by TTP. IL-10 mRNA levels were found to be elevated because of a reduced decay rate in primary macrophages from TTP(-/-) mice. Our study demonstrates the importance of experimental approaches for identifying targets of RNA-binding proteins.