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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.

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 emergence of small open reading frame (sORF)-encoded peptides (SEPs) is rapidly expanding the known proteome at the lower end of the size distribution1,2. Here, we show that the mitochondria proteome is enriched for proteins smaller than 100 a.a. (defined as SEPs). Using a mitochondrial prediction and validation pipeline for small open-reading-frame (sORF)-encoded peptides (SEPs), we report the discovery of 16 endogenous mitochondrial SEPs (mito-SEPs) associated with oxidative phosphorylation (OXPHOS). Through functional prediction, proteomics, metabolomics and metabolic flux modeling, we demonstrate that BRAWNIN (BR), a 71 amino acid peptide encoded by theC12orf73gene, is essential for respiratory chain complex III (CIII) assembly. In human cells, BR is induced by the energy-sensing AMPK pathway, and its depletion impairs mitochondrial ATP production.In vivo, BR is enriched in muscle tissues and its maternal zygotic deletion in zebrafish causes complete CIII loss, resulting in severe growth retardation, lactic acidosis and early death. Our findings demonstrate that BR is essential for oxidative phosphorylation across vertebrate species. We propose that mito-SEPs are an untapped resource for essential regulators of oxidative metabolism.

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.

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.

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 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.

The repression of telomerase activity during cellular differentiation promotes replicative aging and functions as a physiological barrier for tumorigenesis in long-lived mammals, including humans. However, the underlying mechanisms remain largely unclear. Here we describe how miR-615-3p represses hTERT expression. mir-615-3p is located in an intron of the HOXC5 gene, a member of the highly conserved homeobox family of transcription factors controlling embryogenesis and development. Unexpectedly, we found that HoxC5 also represses hTERT expression by disrupting the long-range interaction between hTERT promoter and its distal enhancer. The 3′UTR of hTERT and its upstream enhancer region are well conserved in long-lived primates. Both mir-615-3p and HOXC5 are activated upon differentiation, which constitute a feed-forward loop that coordinates transcriptional and post-transcriptional repression of hTERT during cellular differentiation. Deregulation of HOXC5 and mir-615-3p expression may contribute to the activation of hTERT in human cancers., The expression of telomerase catalytic subunit hTERT is frequently upregulated in many cancers. Here, the authors show HoxC5 and miR-615-3p can negatively regulate hTERT to impede tumorigenesis by targeting the newly evolved cis-regulatory genomic elements of hTERT.

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.

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

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

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 ...

Transcript-specific translational control is generally directed by binding of trans -acting proteins to structural elements in the untranslated region (UTR) of the target mRNA. Here, we elucidate a translational silencing mechanism involving regulated release of an integral ribosomal protein and subsequent binding to its target mRNA. Human ribosomal protein L13a was identified as a candidate interferon- G amma- A ctivated I nhibitor of T ranslation (GAIT) of ceruloplasmin (Cp) mRNA by a genetic screen for Cp 3′-UTR binding proteins. In vitro activity of L13a was shown by inhibition of target mRNA translation by recombinant protein. In response to interferon-γ in vivo, the entire cellular pool of L13a was phosphorylated and released from the 60S ribosomal subunit. Released L13a specifically bound the 3′-UTR GAIT element of Cp mRNA and silenced translation. We propose a model in which the ribosome functions not only as a protein synthesis machine, but also as a depot for regulatory proteins that modulate translation.

SummaryStem cell differentiation involves changes in transcription, but little is known about translational control during differentiation. We comprehensively profiled gene expression during differentiation of murine embryonic stem cells (ESCs) into embryoid bodies by integrating transcriptome analysis with global assessment of ribosome loading. While protein synthesis was parsimonious during self-renewal, differentiation induced an anabolic switch, with global increases in transcript abundance, polysome content, protein synthesis, and protein content. Furthermore, 78% of transcripts showed increased ribosome loading, thereby enhancing translational efficiency. Transcripts under exclusive translational control included the transcription factor ATF5, the tumor suppressor DCC, and the β-catenin agonist Wnt1. We show that a hierarchy of translational regulators, including mTOR, 4EBP1, and the RNA-binding proteins DAZL and GRSF1, control global and selective protein synthesis during ESC differentiation. Parsimonious translation in pluripotent state and hierarchical translational regulation during differentiation may be important quality controls for self-renewal and choice of fate in ESCs.