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Skin homeostasis is indispensable for terrestrial life. The skin, sitting at the interface between the dry external environment and the aqueous interior, acts as a barrier to delimit each from the other. Maintaining this barrier function is critical – if it is compromised, pathogen ingress and water loss may have potentially severe consequences. Skin function is supported by an intricate regulatory network incorporating both protein-coding genes as well as non-coding RNA transcripts. This review will focus on microRNAs, a subset of non-coding RNAs which target specific messenger RNAs for post-transcriptional repression. MicroRNAs regulate every aspect of skin biology, from initial cell fate determination during embryogenesis, to steady state keratinocyte maturation and barrier renewal, as well as coordination of the healing process in response to skin damage. An increased appreciation for the role of microRNAs in skin pathologies not only sheds light on the importance of these small RNAs in healthy skin function, but also provides an expanding repertoire of potential therapeutic targets through which these conditions may be addressed. Mammalian skin appears simple. Its core function is to shield the body from the external environment. Water must be retained to sustain life, whilst harmful pathogens and other environmental insults must be kept out. This simplicity in function belies the array of intricate processes necessary to maintain the skin’s barrier function – multiple series of highly-ordered molecular and cellular events control constant epidermal rejuvenation, as well as rapid repair of any epithelial damage sustained. For internal homeostasis to be maintained, epithelial integrity is essential, and contingent upon precise spatiotemporal control of protein expression in the skin.

Squamous cell carcinoma (SCC) is a lethal malignancy with a high propensity for metastasis. Follistatin-like 1 (FSTL1), a pro-metastatic glycoprotein, is absent from healthy epithelia and aberrantly upregulated in SCC. The FSTL1 transcript encodes two alternative gene products whose dominance is post-transcriptionally regulated via a bistable switch. In healthy epithelia, FSTL1 mRNA is destabilized by binding of KH-type splicing regulatory protein (KSRP), and processed as a primary microRNA encoding miR-198. In SCC, KSRP downregulation terminates miR-198 processing, enabling FSTL1 translation. Here, we identify HuR (Human Antigen R) as an upstream regulator of FSTL1 and describe how downregulation of KSRP is permissive, but not sufficient, to promote sustained FSTL1 expression. Moreover, we demonstrate how the interplay between two RNA-binding proteins controls the translation of pro-oncogenic FSTL1. Increased expression of HuR in SCC outcompetes KSRP and enhances FSTL1 transcript stability, enabling persistent FSTL1 expression and activation of downstream metastatic pathways.

Atopic dermatitis (AD) is a highly prevalent chronic inflammatory skin disease that is characterized by intense pruritus, seriously affecting patients' quality of life. Its pathophysiology, which involves both the adaptive and innate immune responses as well as skin barrier defects, is still poorly understood. We recently identified a microRNA, miR-335, as a key driver of keratinocyte differentiation and cornification, which is essential for the establishment of a healthy skin barrier. However, expression of miR-335 is lost in AD, leading to barrier defect. We further demonstrated how belinostat, a histone deacetylase inhibitor, can effectively restore miR-335 and resolve the barrier defect in a dry skin model. Here, in this commentary, we highlight the role of belinostat in the treatment of AD and discuss the need for more research into crosstalk between epigenetic and non-coding RNA-based regulation, as well as possible therapeutic strategies targeting the epigenome.

The study of translational regulation requires reliable measurement of both mRNA levels and protein synthesis. Cytoplasmic polyadenylation is a prevalent mode of translational regulation during oogenesis and early embryogenesis. Here the length of the poly(A) tail of an mRNA is coupled to its translatability. We describe a protocol to identify translationally regulated genes and measure their translation rate in the early zebrafish embryo using genome-wide polysome profiling. This protocol relies on the isolation of mRNA by means of an rRNA depletion strategy, which avoids capture bias due to short poly(A) tail that can occur when using conventional oligo(dT)-based methods. We also present a simple PCR-based method to measure the poly(A) tail length of selected mRNAs.

The study of translational regulation requires reliable measurement of both mRNA levels and protein synthesis. Cytoplasmic polyadenylation is a prevalent mode of translational regulation during oogenesis and early embryogenesis. Here the length of the poly(A) tail of an mRNA is coupled to its translatability. We describe a protocol to identify translationally regulated genes and measure their translation rate in the early zebrafish embryo using genome-wide polysome profiling. This protocol relies on the isolation of mRNA by means of an rRNA depletion strategy, which avoids capture bias due to short poly(A) tail that can occur when using conventional oligo(dT)-based methods. We also present a simple PCR-based method to measure the poly(A) tail length of selected mRNAs.

Complex multicellular organisms have evolved sophisticated mechanisms to rapidly resolve epithelial injuries. Epithelial integrity is critical to maintaining internal homeostasis. An epithelial breach represents the potential for pathogen ingress and fluid loss, both of which may have severe consequences if not limited. The mammalian wound healing response involves a finely tuned, self-limiting series of cellular and molecular events orchestrated by the transient activation of specific signalling pathways. Accurate regulation of these events is essential; failure to initiate key steps at the right time delays healing and leads to chronic wounds, while aberrant initiation of wound healing processes may produce cell behaviours that promote cancer progression. In this review, we discuss how wound healing pathways co-opted in cancer lose their stringent regulation and become compromised in their reversibility. We hypothesize on how the commandeering of wound healing 'master regulators' is involved in this process, and also highlight the implications of these findings in the treatment of both chronic wounds and cancer.

MicroRNA-138 (miR-138) is a pro-survival oncomiR for glioma stem cells. In malignant gliomas, dysregulated expression of microRNAs, such as miR-138, promotes Tumour initiation and progression. Here, we identify the ancillary role of the CCAAT/enhancer binding protein β (C/EBPβ) as a transcriptional activator of miR-138. We demonstrate that a short 158 bp DNA sequence encoding the precursor of miR-138-2 is essential and sufficient for transcription of miR-138. This short sequence includes the A-box and B-box elements characteristic of RNA Polymerase III (Pol III) promoters, and is also directly bound by C/EBPβ via an embedded ‘C/EBPβ responsive element’ (CRE). CRE and the Pol III B-box element overlap, suggesting that C/EBPβ and transcription factor 3C (TFIIIC) interact at the miR-138-2 locus. We propose that this interaction is essential for the recruitment of the RNA Pol III initiation complex and associated transcription of the oncomiR, miR-138 in malignant gliomas.

Exploring the parallels between wound healing and epithelial cancers, Sundaram et al. elucidate the mechanism by which cancer cells hijack the wound healing switch to enhance invasion and metastasis in head and neck squamous cell carcinoma., Epithelial carcinomas are well known to activate a prolonged wound-healing program that promotes malignant transformation. Wound closure requires the activation of keratinocyte migration via a dual-state molecular switch. This switch involves production of either the anti-migratory microRNA miR-198 or the pro-migratory follistatin-like 1 (FSTL1) protein from a single transcript; miR-198 expression in healthy skin is down-regulated in favor of FSTL1 upon wounding, which enhances keratinocyte migration and promotes re-epithelialization. Here, we reveal a defective molecular switch in head and neck squamous cell carcinoma (HNSCC). This defect shuts off miR-198 expression in favor of sustained FSTL1 translation, driving metastasis through dual parallel pathways involving DIAPH1 and FSTL1. DIAPH1, a miR-198 target, enhances directional migration through sequestration of Arpin, a competitive inhibitor of Arp2/3 complex. FSTL1 blocks Wnt7a-mediated repression of extracellular signal–regulated kinase phosphorylation, enabling production of MMP9, which degrades the extracellular matrix and facilitates metastasis. The prognostic significance of the FSTL1-DIAPH1 gene pair makes it an attractive target for therapeutic intervention.

Breast cancer manifests as a spectrum of subtypes with distinct molecular signatures, and different responses to treatment. Of these subtypes, triple-negative breast cancer (TNBC) has the worst prognoses and limited therapeutic options. Here we report aberrant expression of microRNA-138 (miR-138) in TNBC. Increased miR-138 expression is highly specific to this subtype, correlates with poor prognosis in patients, and is functionally relevant to cancer progression. Our findings establish miR-138 as a specific diagnostic and prognostic biomarker for TNBC. OncomiR-138 is pro-survival; sequence-specific miR-138 inhibition blocks proliferation, promotes apoptosis and inhibits tumour growth in-vivo. miR-138 directly targets a suite of pro-apoptotic and tumour suppressive genes, including tumour suppressor candidate 2 (TUSC2). miR-138 silences TUSC2 by binding to a unique 5′-UTR target-site, which overlaps with the translation start-site of the transcript. Over-expression of TUSC2 mimics the phenotype of miR-138 knockdown and functional rescue experiments confirm that TUSC2 is a direct downstream target of miR-138. Our report of miR-138 as an oncogenic driver in TNBC, positions it as a viable target for oligonucleotide therapeutics and we envision the potential value of using antimiR-138 as an adjuvant therapy to alleviate this therapeutically intractable cancer.

The emergence of small open reading frame (sORF)-encoded peptides (SEPs) is rapidly expanding the known proteome at the lower end of the size distribution. Here, we show that the mitochondrial proteome, particularly the respiratory chain, is enriched for small proteins. Using a prediction and validation pipeline for SEPs, we report the discovery of 16 endogenous nuclear encoded, mitochondrial-localized SEPs (mito-SEPs). Through functional prediction, proteomics, metabolomics and metabolic flux modeling, we demonstrate that BRAWNIN, a 71 a.a. peptide encoded by C12orf73, is essential for respiratory chain complex III (CIII) assembly. In human cells, BRAWNIN is induced by the energy-sensing AMPK pathway, and its depletion impairs mitochondrial ATP production. In zebrafish, Brawnin deletion causes complete CIII loss, resulting in severe growth retardation, lactic acidosis and early death. Our findings demonstrate that BRAWNIN is essential for vertebrate oxidative phosphorylation. We propose that mito-SEPs are an untapped resource for essential regulators of oxidative metabolism., Small open reading frame-encoded peptides (SEPs), shorter than 100 amino acids, are involved in many cell biological processes. Here the authors identify 16 nuclear-encoded mitochondrial SEPs, including BRAWNIN, an essential regulator of respiratory chain complex III assembly and ATP production.

Background Atopic dermatitis (AD) is a common chronic inflammatory skin disease. Skin barrier defects contribute to disease initiation and development; however, underlying mechanisms remain elusive. Objective To understand the underlying cause of barrier defect, we investigated aberrant expression of specific microRNAs (miRNAs) in AD. Delineating the molecular mechanism of dysregulated miRNA network, we focused on identification of specific drugs that can modulate miRNA expression and repair the defective barrier in AD. Methods A screen for differentially expressed miRNAs between healthy skin and AD lesional skin resulted in the identification of miR-335 as the most consistently downregulated miRNA in AD. Using in silico prediction combined with experimental validation, we characterized downstream miR-335 targets and elucidated the molecular pathways by which this microRNA maintains epidermal homeostasis in healthy skin. Results miR-335 was identified as a potent inducer of keratinocyte differentiation; it exerts this effect by directly repressing SOX6. By recruiting SMARCA complex components, SOX6 suppresses epidermal differentiation and epigenetically silences critical genes involved in keratinocyte differentiation. In AD lesional skin, miR-335 expression is aberrantly lost. SOX6 is abnormally expressed throughout the epidermis, where it impairs skin barrier development. We demonstrate that miR-335 is epigenetically regulated by histone deacetylases; a screen for suitable histone deacetylase inhibitors identified belinostat as a candidate drug that can restore epidermal miR-335 expression and rescue the defective skin barrier in AD. Conclusion Belinostat is of clinical significance not only as a candidate drug for AD treatment, but also as a potential means of stopping the atopic march and further progression of this systemic allergic disease.

Background Commitment of pluripotent stem cells into differentiated cells and associated gene expression necessitate specific epigenetic mechanisms that modify the DNA and corresponding histone proteins to render the chromatin in an open or closed state. This in turn dictates the associated genetic machinery, including transcription factors, acknowledging the cellular signals provided. Activating histone methyltransferases represent crucial enzymes in the epigenetic machinery that cause transcription initiation by delivering the methyl mark on histone proteins. A number of studies have evidenced the vital role of one such histone modifier, DOT1L, in transcriptional regulation. Involvement of DOT1L in differentiating pluripotent human embryonic stem (hES) cells into the cardiac lineage has not yet been investigated. Methods The study was conducted on in-house derived (KIND1) and commercially available (HES3) human embryonic stem cell lines. Chromatin immunoprecipitation (ChIP) was performed followed by sequencing to uncover the cardiac genes harboring the DOT1L specific mark H3K79me2. Following this, dual immunofluorescence was employed to show the DOT1L co-occupancy along with the cardiac progenitor specific marker. DOT1L was knocked down by siRNA to further confirm its role during cardiac differentiation. Results ChIP sequencing revealed a significant number of peaks characterizing H3K79me2 occupancy in the proximity of the transcription start site. This included genes like MYOF, NR2F2, NKX2.5, and HAND1 in cardiac progenitors and cardiomyocytes, and POU5F1 and NANOG in pluripotent hES cells. Consistent with this observation, we also show that DOT1L co-localizes with the master cardiac transcription factor NKX2.5, suggesting its direct involvement during gene activation. Knockdown of DOT1L did not alter the pluripotency of hES cells, but it led to the disruption of cardiac differentiation observed morphologically as well as at transcript and protein levels. Conclusions Collectively, our data suggests the crucial role of H3K79me2 methyltransferase DOT1L for activation of NKX2.5 during the cardiac differentiation of hES cells. Electronic supplementary material The online version of this article (10.1186/s13287-018-0810-8) contains supplementary material, which is available to authorized users.

Human embryonic (hES) stem cells are widely used as an in vitro model to understand global genetic and epigenetic changes that occur during early embryonic development. In-house derived hES cells (KIND1) were subjected to directed differentiation into cardiovascular progenitors (D12) and beating cardiomyocytes (D20). Transcriptome profiling of undifferentiated (D0) and differentiated (D12 and 20) cells was undertaken by microarray analysis. ChIP and sequential ChIP were employed to study role of transcription factor NR2F2 during hES cells differentiation. Microarray profiling showed that an alteration of about 1400 and 1900 transcripts occurred on D12 and D20 respectively compared to D0 whereas only 19 genes were altered between D12 and D20. This was found associated with corresponding expression pattern of chromatin remodelers, histone modifiers, miRNAs and lncRNAs marking the formation of progenitors and cardiomyocytes on D12 and D20 respectively. ChIP sequencing and sequential ChIP revealed the binding of NR2F2 with polycomb group member EZH2 and pluripotent factor OCT4 indicating its crucial involvement in cardiac differentiation. The study provides a detailed insight into genetic and epigenetic changes associated with hES cells differentiation into cardiac cells and a role for NR2F2 is deciphered for the first time to down-regulate OCT-4 via EZH2 during cardiac differentiation.

In the earliest stages of animal development following fertilization, maternally deposited mRNAs direct biological processes to the point of zygotic genome activation (ZGA). These maternal mRNAs undergo cytoplasmic polyadenylation (CPA), suggesting translational control of their activation. To elucidate the biological role of CPA during embryogenesis, we performed genome-wide polysome profiling at several stages of zebrafish development. Our analysis revealed a correlation between CPA and polysome-association dynamics, demonstrating a coupling of translation to the CPA of maternal mRNAs. Pan-embryonic CPA inhibition disrupted the maternal-to-zygotic transition (MZT), causing a failure of developmental progression beyond the mid-blastula transition and changes in global gene expression that indicated a failure of ZGA and maternal mRNA clearance. Among the genes that were differentially expressed were those encoding chromatin modifiers and key transcription factors involved in ZGA, including nanog, pou5f3 and sox19b, which have distinct CPA dynamics. Our results establish the necessity of CPA for ensuring progression of the MZT. The RNA-seq data generated in this study represent a valuable zebrafish resource for the discovery of novel elements of the early embryonic transcriptome.

Targeting EGFR is a validated approach in the treatment of squamous-cell cancers (SCCs), although there are no established biomarkers for predicting response. We have identified a synonymous mutation in EGFR, c.2361G>A (encoding p.Gln787Gln), in two patients with head and neck SCC (HNSCC) who were exceptional responders to gefitinib, and we showed in patient-derived cultures that the A/A genotype was associated with greater sensitivity to tyrosine kinase inhibitors (TKIs) as compared to the G/A and G/G genotypes. Remarkably, single-copy G>A nucleotide editing in isogenic models conferred a 70-fold increase in sensitivity due to decreased stability of the EGFR-AS1 long noncoding RNA (lncRNA). In the appropriate context, sensitivity could be recapitulated through EGFR-AS1 knockdown in vitro and in vivo, whereas overexpression was sufficient to induce resistance to TKIs. Reduced EGFR-AS1 levels shifted splicing toward EGFR isoform D, leading to ligand-mediated pathway activation. In co-clinical trials involving patients and patient-derived xenograft (PDX) models, tumor shrinkage was most pronounced in the context of the A/A genotype for EGFR-Q787Q, low expression of EGFR-AS1 and high expression of EGFR isoform D. Our study reveals how a 'silent' mutation influences the levels of a lncRNA, resulting in noncanonical EGFR addiction, and delineates a new predictive biomarker suite for response to EGFR TKIs.

Kindlins are FERM-containing cytoplasmic proteins that regulate integrin-mediated cell-cell and cell-extracellular matrix (ECM) attachments. Kindlin-3 is expressed in hematopoietic cells, platelets, and endothelial cells. Studies have shown that kindlin-3 stabilizes cell adhesion mediated by ß1, ß2, and ß3 integrins. Apart from integrin cytoplasmic tails, kindlins are known to interact with other cytoplasmic proteins. Here we demonstrate that kindlin-3 can associate with ribosome via the receptor for activated-C kinase 1 (RACK1) scaffold protein based on immunoprecipitation, ribosome binding, and proximity ligation assays. We show that kindlin-3 regulates c-Myc protein expression in the human chronic myeloid leukemia cell line K562. Cell proliferation was reduced following siRNA reduction of kindlin-3 expression and a significant reduction in tumor mass was observed in xenograft experiments. Mechanistically, kindlin-3 is involved in integrin α5ß1-Akt-mTOR-p70S6K signaling; however, its regulation of c-Myc protein expression could be independent of this signaling axis. NRF (Natl Research Foundation, S’pore) ASTAR (Agency for Sci., Tech. and Research, S’pore) MOE (Min. of Education, S’pore) Published version

Aminoacyl tRNA synthetases (ARS) catalyze the ligation of amino acids to cognate tRNAs. Chordate ARSs have evolved distinctive features absent from ancestral forms, including compartmentalization in a multisynthetase complex (MSC), noncatalytic peptide appendages, and ancillary functions unrelated to aminoacylation. Here, we show that glutamyl-prolyl-tRNA synthetase (GluProRS), a bifunctional ARS of the MSC, has a regulated, noncanonical activity that blocks synthesis of a specific protein. GluProRS was identified as a component of the interferon (IFN)-gamma-activated inhibitor of translation (GAIT) complex by RNA affinity chromatography using the ceruloplasmin (Cp) GAIT element as ligand. In response to IFN-gamma, GluProRS is phosphorylated and released from the MSC, binds the Cp 3'-untranslated region in an mRNP containing three additional proteins, and silences Cp mRNA translation. Thus, GluProRS has divergent functions in protein synthesis: in the MSC, its aminoacylation activity supports global translation, but translocation of GluProRS to an inflammation-responsive mRNP causes gene-specific translational silencing.

In addition to the integration of internal and external stimuli that are important for the maintenance of stem cell self-renewal, a regulatory layer, microRNA-mediated translational control, either reinforces or inhibits the self-renewal program. Adult stem cells derived from tissues like muscle, blood, or nerves can self-renew only for limited generations. However, embryonic stem cells have an innate program for self-replication that does not require extrinsic signaling, although miRNAs regulate the decision of whether they make a copy of themselves or differentiate into specialized tissues.

Post-transcriptional control of mRNA is a vital step in the regulation of gene expression. Highly specific combinations of RNA-binding–proteins (RBPs) and microRNAs conjointly contribute to the complexity of post-transcriptional control of mRNAs. Post-transcriptional switches regulated by RBPs control context-specific expression of two alternative gene products from a single transcript.

This unit describes a protocol for genome-wide identification of translationally regulated genes during embryonic stem cell differentiation using integrated transcriptome and translation state profiling. Actively translated mRNAs associated with multiple ribosomes (known as polysomes) and translationally inactive mRNAs sequestered in messenger ribonucleoprotein particles (mRNPs), can be separated by sucrose gradient fractionation based on size. Because the number of ribosomes on a transcript correlates with the rate of synthesis of its encoded protein, this allows an operational distinction between well-translated and poorly translated mRNA molecules. In this analysis, fractionated mRNA and total RNA are used to probe microarrays to identify differentially translated genes. Curr. Protoc. Stem Cell Biol. 18:1B.8.1-1B.8.13. © 2011 by John Wiley & Sons, Inc. Keywords: embryonic stem cells; translational control; polysome analysis

Translational control is a common regulatory mechanism for the expression of iron-related proteins. For example, three enzymes involved in erythrocyte development are regulated by three different control mechanisms: globin synthesis is modulated by heme-regulated translational inhibitor; erythroid 5-aminolevulinate synthase translation is inhibited by binding of the iron regulatory protein to the iron response element in the 5'-untranslated region (UTR); and 15-lipoxygenase is regulated by specific proteins binding to the 3'-UTR. Ceruloplasmin (Cp) is a multi-functional, copper protein made primarily by the liver and by activated macrophages. Cp has important roles in iron homeostasis and in inflammation. Its role in iron metabolism was originally proposed because of its ferroxidase activity and because of its ability to stimulate iron loading into apo-transferrin and iron efflux from liver. We have shown that Cp mRNA is induced by interferon (IFN)-γ in U937 monocytic cells, but synthesis of Cp protein is halted by translational silencing. The silencing mechanism requires binding of a cytosolic inhibitor complex, IFN-Gamma-Activated Inhibitor of Translation (GAIT), to a specific GAIT element in the Cp 3'-UTR. Here, we describe our studies that define and characterize the GAIT element and elucidate the specific trans-acting proteins that bind the GAIT element. Our experiments describe a new mechanism of translational control of an iron-related protein and may shed light on the role that macrophage-derived Cp plays at the intersection of iron homeostasis and inflammation.

Ceruloplasmin (Cp) is a copper protein with important functions in iron homeostasis and in inflammation. Cp mRNA expression is induced by interferon (IFN)-g in U937 monocytic cells, but synthesis of Cp protein is halted after about 12 h by transcript-specific translational silencing. The silencing mechanism requires binding of a 4-component cytosolic inhibitor complex, IFN-gamma-activated inhibitor of translation (GAIT), to a defined structural element (GAIT element) in the Cp 3'-UTR. Translational silencing of Cp mRNA requires the essential proteins of mRNA circularization, suggesting that the translational inhibition requires end-to-end mRNA closure. These studies describe a new mechanism of translational control, and may shed light on the role that macrophage-derived Cp plays at the intersection of iron homeostasis and inflammation.

Aminoacyl tRNA synthetases (ARS) catalyze the ligation of amino acids to cognate tRNAs. Chordate ARSs have evolved distinctive features absent from ancestral forms, including compartmentalization in a multisynthetase complex (MSC), noncatalytic peptide appendages, and ancillary functions unrelated to aminoacylation. Here, we show that glutamyl-prolyl-tRNA synthetase (GluProRS), a bifunctional ARS of the MSC, has a regulated, noncanonical activity that blocks synthesis of a specific protein. GluProRS was identified as a component of the interferon (IFN)-gamma-activated inhibitor of translation (GAIT) complex by RNA affinity chromatography using the ceruloplasmin (Cp) GAIT element as ligand. In response to IFN-gamma, GluProRS is phosphorylated and released from the MSC, binds the Cp 3'-untranslated region in an mRNP containing three additional proteins, and silences Cp mRNA translation. Thus, GluProRS has divergent functions in protein synthesis: in the MSC, its aminoacylation activity supports global translation, but translocation of GluProRS to an inflammation-responsive mRNP causes gene-specific translational silencing.

Ceruloplasmin (Cp) is a multifunctional, copper-containing glycoprotein produced by the liver and secreted into the plasma. As an acute-phase protein, its plasma concentration increases up to twofold during multiple inflammatory conditions. Plasma Cp has been reported to be an independent risk factor for cardiovascular disease, including atherosclerosis, carotid restenosis after endarterectomy, and myocardial infarction (24, 36). Several laboratories have shown that Cp copper can cause oxidative modification of low density lipoprotein, and this activity may contribute to a direct role of Cp in the pathogenesis of atherosclerosis (7, 26, 39). Cp also has a ferroxidase activity thought to be necessary for optimal loading of iron into transferrin (27). The important physiological role for Cp in iron homeostasis has been convincingly demonstrated by finding iron overload in humans with hereditary Cp deficiency (40) and in mice with targeted Cp gene disruption (9). In addition to its synthesis by hepatic cells, Cp is synthesized and secreted by activated monocyte/macrophages. Treatment of human monocytic U937 cells or peripheral blood monocytes with gamma interferon (IFN-γ) induces the expression of both Cp mRNA and protein (19). Our laboratory has shown that induced expression of Cp by IFN-γ is subject to a unique transcript-selective translational silencing mechanism in which synthesis is terminated after about 16 h, even in the presence of abundant Cp mRNA (18). Our results suggest the presence of a cytosolic inhibitor, since lysates from U937 cells treated with IFN-γ for 24 h (but not for 8 h) inhibited in vitro translation of Cp in a reticulocyte lysate. The inhibition was accompanied by the binding of a trans-acting factor to the 247-nucleotide (nt), Cp 3′ untranslated region (3′-UTR), as shown by an RNA electrophoretic mobility shift assay (EMSA) using radiolabeled Cp 3′-UTR as probe and by an in vitro translation assay in which unlabeled Cp 3′-UTR, added in excess as a decoy, overcame the inhibition by cytosolic extracts (18). There are multiple examples of translational regulation directed by proteins interacting with the 3′-UTR (see references 32 and 34 for review). The mechanism by which 3′-UTR-binding proteins inhibit translation-initiation at the distant 5′-UTR is incompletely understood, but studies showing that mRNA may form a closed loop by 5′-to-3′ interactions may provide an important clue (10). This “circular” model has been helpful in understanding 3′-UTR-mediated translational silencing of Cp by IFN-γ (21). The mechanism of translational silencing was investigated by in vitro translation of a heterologous reporter transcript consisting of the luciferase (Luc) open reading frame (ORF) ahead of the full-length Cp 3′-UTR and a 30-nt poly(A) tail. Removal or inactivation of any of the components of transcript circularization, i.e., the poly(A) tail, eukaryotic initiation factor 4G, or poly(A)-binding protein (PABP), prevented the translational silencing activity (21). These results have led us to propose a mechanism of translational control in which interactions of the termini carry a 3′-interacting inhibitor protein (or complex) into the vicinity of the translation-initiation site where it can silence translation, possibly by binding to or interfering with an initiation factor or ribosomal protein (20, 21). Transcript-selective translational control generally is directed by a specific cis element characterized by a requirement for specific structural features as well as for invariant sequences. In our previous study, a limited deletion analysis of the Cp 3′-UTR showed that the binding site of the translational inhibitor was present in overlapping UTR fragments 51-247 and 1-150 [where position 1 is the first nucleotide after the stop codon and 247 is the last nucleotide before the poly(A) tail in the short form of the Cp 3′-UTR] (18). This result suggested that the required element was contained in the UTR region 51-150 common to both fragments. Computational folding of the full-length Cp 3′-UTR indicates that the element is located within an elongated structure comprising multiple stems and loops (see Fig. ​Fig.1A).1A). We here describe a detailed analysis of the Cp 3′-UTR and provide evidence for a novel structural element that mediates the inhibition of Cp translation in IFN-γ-activated monocytic cells. FIG. 1. Location of the translational silencing element in the human Cp 3′-UTR. (A) Folding structure of the full-length Cp 3′-UTR as determined by the mfold algorithm. The terminal positions of key constructs are indicated by boxes. (B) A summary ...