Coding region polyadenylation generates a truncated tRNA synthetase that counters translation repression.

Posttranscriptional regulatory mechanisms superimpose "fine-tuning" control upon "on-off" switches characteristic of gene transcription. We have exploited computational modeling with experimental validation to resolve an anomalous relationship between mRNA expression and protein synthesis. The GAIT (gamma-interferon-activated inhibitor of translation) complex repressed VEGF-A synthesis to a low, constant rate independent of VEGF-A mRNA expression levels. Dynamic model simulations predicted an inhibitory GAIT-element-interacting factor to account for this relationship and led to the identification of a truncated form of glutamyl-prolyl tRNA synthetase (EPRS), a GAIT constituent that mediates binding to target transcripts. The truncated protein, EPRS(N1), shields GAIT-element-bearing transcripts from the inhibitory GAIT complex, thereby dictating a "translational trickle" of GAIT target proteins. EPRS(N1) mRNA is generated by polyadenylation-directed conversion of a Tyr codon in the EPRS-coding sequence to a stop codon (PAY(∗)). Genome-wide analysis revealed multiple candidate PAY(∗) targets, including the authenticated target RRM1, suggesting a general mechanism for production of C terminus-truncated regulatory proteins.

Cooperation and competition by RNA-binding proteins in cancer.

Post-transcriptional regulation of gene expression plays a major role in determining the cellular proteome in health and disease. Post-transcriptional control mechanisms are disrupted in many cancers, contributing to multiple processes of tumorigenesis. RNA-binding proteins (RBPs), the main post-transcriptional regulators, often show altered expression and activity in cancer cells. Dysregulation of RBPs contributes to many cancer phenotypes, functioning in complex regulatory networks with other cellular players such as non-coding RNAs, signaling mediators and transcription factors to alter the expression of oncogenes and tumor suppressor genes. RBPs often function combinatorially, based on their binding to target sequences/structures on shared mRNA targets, to regulate the expression of cancer-related genes. This gives rise to cooperativity and competition between RBPs in mRNA binding and resultant functional outcomes in post-transcriptional processes such as mRNA splicing, stability, export and translation. Cooperation and competition is also observed in the case of interaction of RBPs and microRNAs with mRNA targets. RNA structural change is a common mechanism mediating the cooperative/competitive interplay between RBPs and between RBPs and microRNAs. RNA modifications, leading to changes in RNA structure, add a new dimension to cooperative/competitive binding of RBPs to mRNAs, further expanding the RBP regulatory landscape. Therefore, cooperative/competitive interplay between RBPs is a major determinant of the RBP interactome and post-transcriptional regulation of gene expression in cancer cells.

Quorum Sensing by Gelsolin Regulates Programmed Cell Death 4 Expression and a Density-Dependent Phenotype in Macrophages.

Quorum-sensing mechanisms that sense the density of immune cells at the site of inflammation to initiate inflammation resolution have recently been demonstrated as a major determinant of the inflammatory response. We observed a density-dependent increase in expression of the inflammatory tumor suppressor protein programmed cell death 4 (PDCD4) in mouse macrophage cells. Conditioned medium from high-density cells upregulated PDCD4 expression, revealing the presence of a secreted factor(s) acting as a macrophage quorum sensor. Secreted gelsolin (GSN) was identified as the quorum-sensing autoinducer. Alteration of GSN levels changed PDCD4 expression and the density-dependent phenotype of cells. LPS induced the expression of microRNA miR-21, which downregulated both GSN and PDCD4 expression, and reversed the high-density phenotype. The high-density phenotype was correlated with an anti-inflammatory gene expression program, which was counteracted by inflammatory stimulus. Together, our observations establish the miR-21-GSN-PDCD4 regulatory network as a crucial mediator of a macrophage quorum-sensing mechanism for the control of inflammatory responses.

RNA-binding proteins La and HuR cooperatively modulate translation repression of PDCD4 mRNA.

Posttranscriptional regulation of gene expression plays a critical role in controlling the inflammatory response. An uncontrolled inflammatory response results in chronic inflammation, often leading to tumorigenesis. Programmed cell death 4 (PDCD4) is a proinflammatory tumor-suppressor gene which helps to prevent the transition from chronic inflammation to cancer. PDCD4 mRNA translation is regulated by an interplay between the oncogenic microRNA miR-21 and the RNA-binding protein (RBP) human antigen R (HuR) in response to lipopolysaccharide stimulation, but the role of other regulatory factors remains unknown. Here, we report that the RBP lupus antigen (La) interacts with the 3'-untranslated region of PDCD4 mRNA and prevents miR-21-mediated translation repression. While lipopolysaccharide causes nuclear-cytoplasmic translocation of HuR, it enhances cellular La expression. Remarkably, La and HuR were found to bind cooperatively to the PDCD4 mRNA and mitigate miR-21-mediated translation repression. The cooperative action of La and HuR reduced cell proliferation and enhanced apoptosis, reversing the pro-oncogenic function of miR-21. Together, these observations demonstrate a cooperative interplay between two RBPs, triggered differentially by the same stimulus, which exerts a synergistic effect on PDCD4 expression and thereby helps maintain a balance between inflammation and tumorigenesis.

Transcriptome-wide analysis reveals spatial correlation between N6-methyladenosine and binding sites of microRNAs and RNA-binding proteins.

N6-methyladenosine (m6A), the most prevalent epitranscriptomic modification in eukaryotes, is enriched in 3'-untranslated regions (3'UTRs) of mRNAs. As 3'UTRs are major binding sites of RNA-binding proteins (RBPs) and microRNAs (miRNAs), m6A-dependent local RNA structure change may alter the accessibility of RBPs and miRNAs to their target sites and regulate mRNA function. Using a human transcriptome-wide computational analysis to investigate the relation between m6A, RBPs and miRNAs, we find a strong positive correlation between number of m6A sites, miRNAs and RBPs binding to mRNAs, suggesting m6A-modified mRNAs are more targeted by miRNAs and RBPs. Moreover, m6A sites are located proximally to miRNA target sites and binding sites of multiple RBPs. Further, miRNA target sites and RBP-binding sites located close to each other are also located proximally to m6A. This study indicates three-way interplay between m6A, microRNA and RBP binding, suggesting the influence of mRNA modifications on the miRNA and RBP interactomes.

Cyclometalated rhodium and iridium complexes with imidazole containing Schiff bases: Synthesis, structure and cellular imaging.

Cyclometalated rhodium(III) and iridium(III) complexes (1-4) of two Schiff base ligands L1 and L2 with the general formula [M(ppy)2(Ln)]Cl {M = Rh, Ir; ppy = 2-phenylpyridine; n = 1, 2; L = Schiff base ligand} have been synthesized. The new ligands and the complexes have been characterized with spectroscopic techniques. Electrochemistry of the complexes revealed anodic behavior, corresponding to an M(III) to M(IV) oxidation. The X-ray crystal structures of complexes 2 and 4 have also been determined to interpret the coordination behavior of the complexes. Photophysical study shows that all the complexes display fluorescence at room temperature with quantum yield of about 3 × 10-2 to 5 × 10-2. The electronic absorption spectra of all the complexes fit well with the computational studies. Cellular imaging studies were done with the newly synthesized complexes. To the best of our knowledge, this is the first report of organometallic complexes of rhodium(III) and iridium(III) with Schiff base ligands explored for cellular imaging. Emphasis of this work lies on the structural features, photophysical behavior, cellular uptake and imaging of the fluorescent transition metal complexes.

The HILDA complex coordinates a conditional switch in the 3'-untranslated region of the VEGFA mRNA.

Cell regulatory circuits integrate diverse, and sometimes conflicting, environmental cues to generate appropriate, condition-dependent responses. Here, we elucidate the components and mechanisms driving a protein-directed RNA switch in the 3'UTR of vascular endothelial growth factor (VEGF)-A. We describe a novel HILDA (hypoxia-inducible hnRNP L-DRBP76-hnRNP A2/B1) complex that coordinates a three-element RNA switch, enabling VEGFA mRNA translation during combined hypoxia and inflammation. In addition to binding the CA-rich element (CARE), heterogeneous nuclear ribonucleoprotein (hnRNP) L regulates switch assembly and function. hnRNP L undergoes two previously unrecognized, condition-dependent posttranslational modifications: IFN-γ induces prolyl hydroxylation and von Hippel-Lindau (VHL)-mediated proteasomal degradation, whereas hypoxia stimulates hnRNP L phosphorylation at Tyr(359), inducing binding to hnRNP A2/B1, which stabilizes the protein. Also, phospho-hnRNP L recruits DRBP76 (double-stranded RNA binding protein 76) to the 3'UTR, where it binds an adjacent AU-rich stem-loop (AUSL) element, "flipping" the RNA switch by disrupting the GAIT (interferon-gamma-activated inhibitor of translation) element, preventing GAIT complex binding, and driving robust VEGFA mRNA translation. The signal-dependent, HILDA complex coordinates the function of a trio of neighboring RNA elements, thereby regulating translation of VEGFA and potentially other mRNA targets. The VEGFA RNA switch might function to ensure appropriate angiogenesis and tissue oxygenation during conflicting signals from combined inflammation and hypoxia. We propose the VEGFA RNA switch as an archetype for signal-activated, protein-directed, multi-element RNA switches that regulate posttranscriptional gene expression in complex environments.

Interplay between RNA-binding protein HuR and microRNA-125b regulates p53 mRNA translation in response to genotoxic stress.

Tumor suppressor protein p53 plays a crucial role in maintaining genomic integrity in response to DNA damage. Regulation of translation of p53 mRNA is a major mode of regulation of p53 expression under genotoxic stress. The AU/U-rich element-binding protein HuR has been shown to bind to p53 mRNA 3'UTR and enhance translation in response to DNA-damaging UVC radiation. On the other hand, the microRNA miR-125b is reported to repress p53 expression and stress-induced apoptosis. Here, we show that UVC radiation causes an increase in miR-125b level in a biphasic manner, as well as nuclear cytoplasmic translocation of HuR. Binding of HuR to the p53 mRNA 3'UTR, especially at a site adjacent to the miR-125b target site, causes dissociation of the p53 mRNA from the RNA-induced silencing complex (RISC) and inhibits the miR-125b-mediated translation repression of p53. HuR prevents the oncogenic effect of miR-125b by reversing the decrease in apoptosis and increase in cell proliferation caused by the overexpression of miR-125b. The antagonistic interplay between miR-125b and HuR might play an important role in fine-tuning p53 gene expression at the post-transcriptional level, and thereby regulate the cellular response to genotoxic stress.

DAPK-ZIPK-L13a axis constitutes a negative-feedback module regulating inflammatory gene expression.

Phosphorylation of ribosomal protein L13a is essential for translational repression of inflammatory genes by the interferon (IFN)-gamma-activated inhibitor of translation (GAIT) complex. Here we show that IFN-gamma activates a kinase cascade in which death-associated protein kinase-1 (DAPK) activates zipper-interacting protein kinase (ZIPK), culminating in L13a phosphorylation on Ser(77), L13a release from the ribosome, and translational silencing of GAIT element-bearing target mRNAs. Remarkably, both kinase mRNAs contain functional 3'UTR GAIT elements, and thus the same inhibitory pathway activated by the kinases is co-opted to suppress their expression. Inhibition of DAPK and ZIPK facilitates cell restoration to the basal state and allows renewed induction of GAIT target transcripts by repeated stimulation. Thus, the DAPK-ZIPK-L13a axis forms a unique regulatory module that first represses, then repermits inflammatory gene expression. We propose that the module presents an important checkpoint in the macrophage "resolution of inflammation" program, and that pathway defects may contribute to chronic inflammatory disorders.

A stress-responsive RNA switch regulates VEGFA expression.

Ligand binding to structural elements in the non-coding regions of messenger RNA modulates gene expression. Ligands such as free metabolites or other small molecules directly bind and induce conformational changes in regulatory RNA elements known as riboswitches. Other types of RNA switches are activated by complexed metabolites-for example, RNA-ligated metabolites such as aminoacyl-charged transfer RNA in the T-box system, or protein-bound metabolites in the glucose- or amino-acid-stimulated terminator-anti-terminator systems. All of these switch types are found in bacteria, fungi and plants. Here we report an RNA switch in human vascular endothelial growth factor-A (VEGFA, also known as VEGF) mRNA 3' untranslated region (UTR) that integrates signals from interferon (IFN)-gamma and hypoxia to regulate VEGFA translation in myeloid cells. Analogous to riboswitches, the VEGFA 3' UTR undergoes a binary conformational change in response to environmental signals. However, the VEGFA 3' UTR switch is metabolite-independent, and the conformational change is dictated by mutually exclusive, stimulus-dependent binding of proteins, namely, the IFN-gamma-activated inhibitor of translation complex and heterogeneous nuclear ribonucleoprotein L (HNRNPL, also known as hnRNP L). We speculate that the VEGFA switch represents the founding member of a family of signal-mediated, protein-dependent RNA switches that evolved to regulate gene expression in multicellular animals in which the precise integration of disparate inputs may be more important than the rapidity of response.

The GAIT system: a gatekeeper of inflammatory gene expression.

Functionally related genes are coregulated by specific RNA-protein interactions that direct transcript-selective translational control. In myeloid cells, interferon (IFN)-gamma induces formation of the heterotetrameric, IFN-gamma-activated inhibitor of translation (GAIT) complex comprising glutamyl-prolyl tRNA synthetase (EPRS), NS1-associated protein 1 (NSAP1), ribosomal protein L13a and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This complex binds defined 3' untranslated region elements within a family of inflammatory mRNAs and suppresses their translation. IFN-gamma-dependent phosphorylation, and consequent release of EPRS and L13a from the tRNA multisynthetase complex and 60S ribosomal subunit, respectively, regulates GAIT complex assembly. EPRS recognizes and binds target mRNAs, NSAP1 negatively regulates RNA binding, and L13a inhibits translation initiation by binding eukaryotic initiation factor 4G. Repression of a post-transcriptional regulon by the GAIT system might contribute to the resolution of chronic inflammation.

Systematic analysis of RNA-binding proteins identifies targetable therapeutic vulnerabilities in osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumor with a strong tendency to metastasize, limiting the prognosis of affected patients. Genomic, epigenomic and transcriptomic analyses have demonstrated the exquisite molecular complexity of this tumor, but have not sufficiently defined the underlying mechanisms or identified promising therapeutic targets. To systematically explore RNA-protein interactions relevant to OS, we define the RNA interactomes together with the full proteome and the transcriptome of cells from five malignant bone tumors (four osteosarcomata and one malignant giant cell tumor of the bone) and from normal mesenchymal stem cells and osteoblasts. These analyses uncover both systematic changes of the RNA-binding activities of defined RNA-binding proteins common to all osteosarcomata and individual alterations that are observed in only a subset of tumors. Functional analyses reveal a particular vulnerability of these tumors to translation inhibition and a positive feedback loop involving the RBP IGF2BP3 and the transcription factor Myc which affects cellular translation and OS cell viability. Our results thus provide insight into potentially clinically relevant RNA-binding protein-dependent mechanisms of osteosarcoma.

p53 and HuR combinatorially control the biphasic dynamics of microRNA-125b in response to genotoxic stress.

Post-transcriptional regulation of p53, by the microRNA miR-125b and the RNA-binding protein HuR, controls p53 expression under genotoxic stress. p53 mRNA translation is repressed by miR-125b, tightly regulating its basal level of expression. The repression is relieved upon DNA damage by a decrease in miR-125b level, contributing to pulsatile expression of p53. The pulse of p53, as also of HuR, in response to UV irradiation coincides with a time-dependent biphasic change in miR-125b level. We show that the cause for the decrease in miR-125b level immediately post DNA-damage is enhanced exosomal export mediated by HuR. The subsequent increase in miR-125b level is due to p53-mediated transcriptional upregulation and enhanced processing, demonstrating miR-125b as a transcriptional and processing target of p53. p53 activates the transcription of primary miR-125b RNA from a cryptic promoter in response to UV irradiation. Together, these regulatory processes constitute reciprocal feedback loops that determine the biphasic change in miR-125b level, ultimately contributing to the fine-tuned temporal regulation of p53 expression in response to genotoxic stress.

An AKT1-and TRIM21-mediated phosphodegron controls proteasomal degradation of HuR enabling cell survival under heat shock.

Post-transcriptional regulation by RNA-binding proteins (RBPs) is a major mode of controlling gene expression under stress conditions. The RBP HuR regulates the translation/turnover of multiple mRNAs in stress responses. HuR is degraded in response to heat stress consequent to ubiquitination of the K182 amino acid residue. We have identified TRIM21 as the E3-ubiquitin ligase causing HuR polyubiquitination at K182 and proteasomal degradation under heat shock. The S100 and E101 residues are required for binding of TRIM21 to HuR. Heat shock-induced phosphorylation of S100 is necessary for TRIM21 interaction with HuR and subsequent degradation. We identified AKT1 as the kinase which phosphorylates S100, allowing the recognition of HuR by TRIM21. Sequential phosphorylation by AKT1 and ubiquitination by TRIM21 therefore determine a "phosphodegron" in HuR that is required for regulating the cellular level of HuR under heat shock, thereby enabling a crucial adaptive mechanism allowing cell survival in response to heat stress.

Fates and functions of RNA-binding proteins under stress.

Exposure to stress activates a well-orchestrated set of changes in gene expression programs that allow the cell to cope with and adapt to the stress, or undergo programmed cell death. RNA-protein interactions, mediating all aspects of post-transcriptional regulation of gene expression, play crucial roles in cellular stress responses. RNA-binding proteins (RBPs), which interact with sequence/structural elements in RNAs to control the steps of RNA metabolism, have therefore emerged as central regulators of post-transcriptional responses to stress. Following exposure to a variety of stresses, the dynamic alterations in the RNA-protein interactome enable cells to respond to intracellular or extracellular perturbations by causing changes in mRNA splicing, polyadenylation, stability, translation, and localization. As RBPs play a central role in determining the cellular proteome both qualitatively and quantitatively, it has become increasingly evident that their abundance, availability, and functions are also highly regulated in response to stress. Exposure to stress initiates a series of signaling cascades that converge on post-translational modifications (PTMs) of RBPs, resulting in changes in their subcellular localization, association with stress granules, extracellular export, proteasomal degradation, and RNA-binding activities. These alterations in the fate and function of RBPs directly impact their post-transcriptional regulatory roles in cells under stress. Adopting the ubiquitous RBP HuR as a prototype, three scenarios illustrating the changes in nuclear-cytoplasmic localization, RNA-binding activity, export and degradation of HuR in response to inflammation, genotoxic stress, and heat shock depict the complex and interlinked regulatory mechanisms that control the fate and functions of RBPs under stress. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Evolution of function of a fused metazoan tRNA synthetase.

The origin and evolution of multidomain proteins are driven by diverse processes including fusion/fission, domain shuffling, and alternative splicing. The 20 aminoacyl-tRNA synthetases (AARS) constitute an ancient conserved family of multidomain proteins. The glutamyl-prolyl tRNA synthetase (EPRS) of bilaterian animals is unique among AARSs, containing two functional enzymes catalyzing ligation of glutamate and proline to their cognate transfer RNAs (tRNAs). The ERS and PRS catalytic domains in multiple bilaterian taxa are linked by variable number of helix-turn-helix domains referred to as WHEP-TRS domains. In addition to its canonical aminoacylation activities, human EPRS exhibits a noncanonical function as an inflammation-responsive regulator of translation. Recently, we have shown that the WHEP domains direct this auxiliary function of human EPRS by interacting with an mRNA stem-loop element (interferon-gamma-activated inhibitor of translation [GAIT] element). Here, we show that EPRS is present in the cnidarian Nematostella vectensis, which pushes the origin of the fused protein back to the cnidarian-bilaterian ancestor, 50-75 My before the origin of the Bilateria. Remarkably, the Nematostella EPRS mRNA is alternatively spliced to yield three isoforms with variable number and sequence of WHEP domains and with distinct RNA-binding activities. Whereas one isoform containing a single WHEP domain binds tRNA, a second binds both tRNA and GAIT element RNA. However, the third isoform contains two WHEP domains and like the human ortholog binds specifically to GAIT element RNA. These results suggest that alternative splicing of WHEP domains in the EPRS gene of the cnidarian-bilaterian ancestor gave rise to a novel molecular function of EPRS conserved during metazoan evolution.

Targeting ribosome assembly on the HCV RNA using a small RNA molecule.

Translation initiation of hepatitis C Virus (HCV) RNA is the initial obligatory step of the viral life cycle, mediated through the Internal Ribosome Entry Site (IRES) present in the 5'-untranslated region (UTR). Initiation on the HCV IRES is mediated by multiple structure-specific interactions between IRES RNA and host 40S ribosomal subunit. In the present study we demonstrate that the SLIIIef domain, in isolation from other structural elements of HCV IRES, retain the ability to interact with 40S ribosome subunit. A small RNA SLRef, mimicking the SLIIIef domain was found to interact specifically with human La protein and the ribosomal protein S5 and selectively inhibit HCV RNA translation. More importantly, SLRef RNA showed significant suppression of replication in HCV monocistronic replicon and decrease of negative strand synthesis in HCV cell culture system. Finally, using Sendai virus based virosome, the targeted delivery of SLRef RNA into mice liver succeeded in selectively inhibiting HCV IRES mediated translation in vivo.

Macromolecular complexes as depots for releasable regulatory proteins.

Multi-component, macromolecular complexes perform essential cellular functions that require spatial or temporal coordination of activities. Complexes also facilitate co-regulation of protein amounts and cellular localization of individual components. We propose a novel function of multi-component complexes as depots for regulatory proteins that, upon release, acquire new auxiliary functions. We further propose that component release is inducible and context-dependent. We describe two cases in which multi-component assemblies - the ribosome and tRNA multi-synthetase complex--function as depots. Both complexes have crucial roles in supporting protein synthesis but they also release regulatory proteins for inflammation-responsive, transcript-specific translational control. Recent evidence indicates that other macromolecular assemblies might be sources for proteins with auxiliary functions, and the depot mechanism might be widespread in nature.

Negative feedback regulation by HuR controls TRIM21 expression and function in response to UV radiation.

The E3 ubiquitin ligase TRIM21 plays a crucial role as a negative regulator of innate immune responses. Recent evidence has also indicated the involvement of TRIM21 in the genotoxic stress response and suppressing tumorigenesis. Our previous work has demonstrated a new function of TRIM21 in inhibiting p53 protein synthesis by degrading the RNA-binding protein HuR in response to UV radiation. This suggested a pro-oncogenic role of TRIM21. In this study, we have shown that TRIM21 enhances the proliferation of MCF7 breast carcinoma cells and counteracts the decrease in cell proliferation and colony formation caused by UV-induced DNA damage. Further, this pro-oncogenic role of TRIM21 in response to DNA damage is mediated by its degradation of HuR. Conversely, we found that HuR binds to a U-rich element in the 3'UTR of TRIM21 mRNA and activates its translation, thereby constituting a negative feedback loop. We found that dihydrotanshinone-I (DHTS-I), a plant-derived product which prevents HuR binding to specific RNAs, prevented HuR-mediated upregulation of TRIM21, while increasing the HuR-mediated upregulation of p53. Together, these findings demonstrate a negative feedback regulation between TRIM21 and HuR, which may play an important role in regulating the level of p53 in the genotoxic stress response.

A cyclometalated trinuclear Ir(iii)/Pt(ii) complex as a luminescent probe for histidine-rich proteins.

Organometallic complexes have important application in the field of protein staining, with potential for use in proteomic analysis. The rational synthesis of a trinuclear luminescent organometallic complex with two platinum(ii) centres appended to the cyclometalated ligand of the iridium(iii) centre is reported here. Two di-2-picolylamine groups bonded to the cyclometalated phenyl pyridine moiety provide three coordinating sites to each platinum centre. The replacement of chloride in the fourth coordination site of two square planar platinum metal centres with the imidazole nitrogen or sulphur atom of histidine/cysteine is evident from the change in luminescence intensity upon binding these amino acids. The increase in luminescence emission intensity upon binding of histidine to the organometallic complex allowed it to be used as a protein staining agent. Reversibility of staining upon washing with imidazole enhances the possibility of its application in mass spectrometric analysis.