lncRNA LINC00941 modulates MTA2/NuRD occupancy to suppress premature human epidermal differentiation.

Numerous long non-coding RNAs (lncRNAs) were shown to have a functional impact on cellular processes such as human epidermal homeostasis. However, the mechanism of action for many lncRNAs remains unclear to date. Here, we report that lncRNA LINC00941 regulates keratinocyte differentiation on an epigenetic level through association with the NuRD complex, one of the major chromatin remodelers in cells. We find that LINC00941 interacts with NuRD-associated MTA2 and CHD4 in human primary keratinocytes. LINC00941 perturbation changes MTA2/NuRD occupancy at bivalent chromatin domains in close proximity to transcriptional regulator genes, including the EGR3 gene coding for a transcription factor regulating epidermal differentiation. Notably, LINC00941 depletion resulted in reduced NuRD occupancy at the EGR3 gene locus, increased EGR3 expression in human primary keratinocytes, and increased abundance of EGR3-regulated epidermal differentiation genes in cells and human organotypic epidermal tissues. Our results therefore indicate a role of LINC00941/NuRD in repressing EGR3 expression in non-differentiated keratinocytes, consequentially preventing premature differentiation of human epidermal tissues.

The human long non-coding RNA LINC00941 and its modes of action in health and disease.

Long non-coding RNAs have gained attention in recent years as they were shown to play crucial roles in the regulation of cellular processes, but the understanding of the exact mechanisms is still incomplete in most cases. This is also true for long non-coding RNA LINC00941, which was recently found to be highly upregulated in various types of cancer influencing cell proliferation and metastasis. Initial studies could not elucidate the mode of action to understand the role and real impact of LINC00941 in tissue homeostasis and cancer development. However, recent analyses have demonstrated multiple potential modes of action of LINC00941 influencing the functionality of various cancer cell types. Correspondingly, LINC00941 was proposed to be involved in regulation of mRNA transcription and modulation of protein stability, respectively. In addition, several experimental approaches suggest a function of LINC00941 as competitive endogenous RNA, thus acting in a post-transcriptional regulatory fashion. This review summarizes our recent knowledge about the mechanisms of action of LINC00941 elucidated so far and discusses its putative role in miRNA sequestering processes. In addition, the functional role of LINC00941 in regulating human keratinocytes is discussed to also highlight its role in normal tissue homeostasis tissue aside from its involvement in cancer.

The Interplay of NEAT1 and miR-339-5p Influences on Mesangial Gene Expression and Function in Various Diabetic-Associated Injury Models.

Mesangial cells (MCs), substantial cells for architecture and function of the glomerular tuft, take a key role in progression of diabetic kidney disease (DKD). Despite long standing researches and the need for novel therapies, the underlying regulatory mechanisms in MCs are elusive. This applies in particular to long non-coding RNAs (lncRNA) but also microRNAs (miRNAs). In this study, we investigated the expression of nuclear paraspeckle assembly transcript 1 (NEAT1), a highly conserved lncRNA, in several diabetes in-vitro models using human MCs. These cells were treated with high glucose, TGFß, TNAα, thapsigargin, or tunicamycin. We analyzed the implication of NEAT1 silencing on mesangial cell migration, proliferation, and cell size as well as on mRNA and miRNA expression. Here, the miRNA hsa-miR-339-5p was not only identified as a potential interaction partner for NEAT1 but also for several coding genes. Furthermore, overexpression of hsa-miR-339-5p leads to a MC phenotype comparable to a NEAT1 knockdown. In-silico analyses also underline a relevant role of NEAT1 and hsa-miR-339-5p in mesangial physiology, especially in the context of DKD.

From structure to function: Route to understanding lncRNA mechanism.

RNAs have emerged as a major target for diagnostics and therapeutics approaches. Regulatory nonprotein-coding RNAs (ncRNAs) in particular display remarkable versatility. They can fold into complex structures and interact with proteins, DNA, and other RNAs, thus modulating activity, localization, or interactome of multi-protein complexes. Thus, ncRNAs confer regulatory plasticity and represent a new layer of regulatory control. Interestingly, long noncoding RNAs (lncRNAs) tend to acquire complex secondary and tertiary structures and their function-in many cases-is dependent on structural conservation rather than primary sequence conservation. Whereas for many proteins, structure and its associated function are closely connected, for lncRNAs, the structural domains that determine functionality and its interactome are still not well understood. Numerous approaches for analyzing the structural configuration of lncRNAs have been developed recently. Here, will provide an overview of major experimental approaches used in the field, and discuss the potential benefit of using combinatorial strategies to analyze lncRNA modes of action based on structural information.

Glomerular expression pattern of long non-coding RNAs in the type 2 diabetes mellitus BTBR mouse model.

The prevalence of type 2 diabetes mellitus (T2DM) and by association diabetic nephropathy (DN) will continuously increase in the next decades. Nevertheless, the underlying molecular mechanisms are largely unknown and studies on the role of new actors like long non-coding RNAs (lncRNAs) barely exist. In the present study, the inherently insulin-resistant mouse strain "black and tan, brachyuric" (BTBR) served as T2DM model. While wild-type mice do not exhibit pathological changes, leptin-deficient diabetic animals develop a severe T2DM accompanied by a DN, which closely resembles the human phenotype. We analyzed the glomerular expression of lncRNAs from wild-type and diabetic BTBR mice (four, eight, 16, and 24 weeks) applying the "GeneChip Mouse Whole Transcriptome 1.0 ST" array. This microarray covered more lncRNA gene loci than any other array before. Over the observed time, our data revealed differential expression patterns of 1746 lncRNAs, which markedly differed from mRNAs. We identified protein-coding and non-coding genes, that were not only co-located but also co-expressed, indicating a potentially cis-acting function of these lncRNAs. In vitro-experiments strongly suggested a cell-specific expression of these lncRNA-mRNA-pairs. Additionally, protein-coding genes, being associated with significantly regulated lncRNAs, were enriched in various biological processes and pathways, that were strongly linked to diabetes.

The long non-coding RNA LINC00941 and SPRR5 are novel regulators of human epidermal homeostasis.

Several long non-coding RNAs (lncRNAs) act as regulators of cellular homeostasis; however, few of these molecules were functionally characterized in a mature human tissue environment. Here, we report that the lncRNA LINC00941 is a crucial regulator of human epidermal homeostasis. LINC00941 is enriched in progenitor keratinocytes and acts as a repressor of keratinocyte differentiation. Furthermore, LINC00941 represses SPRR5, a previously uncharacterized molecule, which functions as an essential positive regulator of keratinocyte differentiation. Interestingly, 54.8% of genes repressed in SPRR5-deficient epidermal tissue are induced in LINC00941-depleted organotypic epidermis, suggesting a common mode of action for both molecules.

Splicing and Chromatin Factors Jointly Regulate Epidermal Differentiation.

Epidermal homeostasis requires balanced progenitor cell proliferation and loss of differentiated cells from the epidermal surface. During this process, cells undergo major changes in their transcriptional programs to accommodate new cellular functions. We found that transcriptional and post-transcriptional mechanisms underlying these changes jointly control genes involved in cell adhesion, a key process in epidermal maintenance. Using siRNA-based perturbation screens, we identified DNA and/or RNA binding regulators of epidermal differentiation. Computational modeling and experimental validation identified functional interactions between the matrin-type 2 zinc-finger protein ZMAT2 and the epigenetic modifiers ING5, SMARCA5, BRD1, UHRF1, BPTF, and SMARCC2. ZMAT2 is an interactor of the pre-spliceosome that is required to keep cells in an undifferentiated, proliferative state. RNA immunoprecipitation and transcriptome-wide RNA splicing analysis showed that ZMAT2 associates with and regulates transcripts involved in cell adhesion in conjunction with ING5. Thus, joint control by splicing regulation, histone, and DNA modification is important to maintain epidermal cells in an undifferentiated state.

Non-coding RNAs: Classification, Biology and Functioning.

One of the long-standing principles of molecular biology is that DNA acts as a template for transcription of messenger RNAs, which serve as blueprints for protein translation. A rapidly growing number of exceptions to this rule have been reported over the past decades: they include long known classes of RNAs involved in translation such as transfer RNAs and ribosomal RNAs, small nuclear RNAs involved in splicing events, and small nucleolar RNAs mainly involved in the modification of other small RNAs, such as ribosomal RNAs and transfer RNAs. More recently, several classes of short regulatory non-coding RNAs, including piwi-associated RNAs, endogenous short-interfering RNAs and microRNAs have been discovered in mammals, which act as key regulators of gene expression in many different cellular pathways and systems. Additionally, the human genome encodes several thousand long non-protein coding RNAs >200 nucleotides in length, some of which play crucial roles in a variety of biological processes such as epigenetic control of chromatin, promoter-specific gene regulation, mRNA stability, X-chromosome inactivation and imprinting. In this chapter, we will introduce several classes of short and long non-coding RNAs, describe their diverse roles in mammalian gene regulation and give examples for known modes of action.

Long non-coding RNA ZFAS1 interacts with CDK1 and is involved in p53-dependent cell cycle control and apoptosis in colorectal cancer.

We determined expression of 83 long non-coding RNAs (lncRNAs) and identified ZFAS1 to be significantly up-regulated in colorectal cancer (CRC) tissue. In cohort of 119 CRC patients we observed that 111 cases displayed at least two-times higher expression of ZFAS1 in CRC compared to paired normal colorectal tissue (P < 0.0001). By use of CRC cell lines (HCT116+/+, HCT116-/- and DLD-1) we showed, that ZFAS1 silencing decreases proliferation through G1-arrest of cell cycle, and also tumorigenicity of CRC cells. We identified Cyclin-dependent kinase 1 (CDK1) as interacting partner of ZFAS1 by pull-down experiment and RNA immunoprecipitation. Further, we have predicted by bioinformatics approach ZFAS1 to sponge miR-590-3p, which was proved to target CDK1. Levels of CDK1 were not affected by ZFAS1 silencing, but cyclin B1 was decreased in both cell lines. We observed significant increase in p53 levels and PARP cleavage in CRC cell lines after ZFAS1 silencing indicating increase in apoptosis. Our data suggest that ZFAS1 may function as oncogene in CRC by two main actions: (i) via destabilization of p53 and through (ii) interaction with CDK1/cyclin B1 complex leading to cell cycle progression and inhibition of apoptosis. However, molecular mechanisms behind these interactions have to be further clarified.

A LncRNA-MAF:MAFB transcription factor network regulates epidermal differentiation.

Progenitor differentiation requires remodeling of genomic expression; however, in many tissues, such as epidermis, the spectrum of remodeled genes and the transcription factors (TFs) that control them are not fully defined. We performed kinetic transcriptome analysis during regeneration of differentiated epidermis and identified gene sets enriched in progenitors (594 genes), in early (159 genes), and in late differentiation (387 genes). Module mapping of 1,046 TFs identified MAF and MAFB as necessary and sufficient for progenitor differentiation. MAF:MAFB regulated 393 genes altered in this setting. Integrative analysis identified ANCR and TINCR lncRNAs as essential upstream MAF:MAFB regulators. ChIP-seq analysis demonstrated MAF:MAFB binding to known epidermal differentiation TF genes whose expression they controlled, including GRHL3, ZNF750, KLF4, and PRDM1. Each of these TFs rescued expression of specific MAF:MAFB target gene subsets in the setting of MAF:MAFB loss, indicating they act downstream of MAF:MAFB. A lncRNA-TF network is thus essential for epidermal differentiation.

RNA binding of PRC2: promiscuous or well ordered?

The polycomb repressive complex 2 (PRC2) methylates histones for epigenetic silencing and associates with thousands of protein-coding and noncoding RNAs. How the recruitment of PRC2 to specific sites is facilitated is currently unclear. Two recent studies have deciphered the impact of RNA binding on PRC2 recruitment and activity (Cifuentes-Rojas et al., 2014; Davidovich et al., 2013).

The non-coding skin: exploring the roles of long non-coding RNAs in epidermal homeostasis and disease.

Long non-coding RNAs (lncRNAs) have recently gained increasing attention because of their crucial roles in gene regulatory processes. Functional studies using mammalian skin as a model system have revealed their role in controlling normal tissue homeostasis as well as the transition to a diseased state. Here, we describe how lncRNAs regulate differentiation to preserve an undifferentiated epidermal progenitor compartment, and to maintain a functional skin permeability barrier. Furthermore, we will reflect on recent work analyzing the impact of lncRNAs on the progression from normal epithelium to the development of skin disorders and cancer.

TINCR, staufen1, and cellular differentiation.

The human genome encodes several thousand long non-protein coding transcripts>200 nucleotides in length, a subset of which were shown to play important roles in regulation of gene expression. We recently identified TINCR, a lncRNA required for induction of key differentiation genes in epidermal tissue, including genes mutated in human skin diseases characterized by disrupted epidermal barrier formation. High-throughput analyses of TINCR RNA- and protein-interactomes revealed TINCR interaction with differentiation mRNAs as well as the Staufen1 protein. TINCR, together with Staufen1, seems to stabilize a subset of mRNAs required for epidermal differentiation. Here, we discuss the emerging roles of Staufen1 and TINCR in the regulation of mammalian cell differentiation mediated by interaction with target mRNAs. We consider a role for TINCR as an epithelial-specific guide for targeting the Staufen1 protein to specific mRNAs, reflecting the increasing complexity of gene regulatory processes in mammalian cells and tissue.

Control of somatic tissue differentiation by the long non-coding RNA TINCR.

Several of the thousands of human long non-coding RNAs (lncRNAs) have been functionally characterized; however, potential roles for lncRNAs in somatic tissue differentiation remain poorly understood. Here we show that a 3.7-kilobase lncRNA, terminal differentiation-induced ncRNA (TINCR), controls human epidermal differentiation by a post-transcriptional mechanism. TINCR is required for high messenger RNA abundance of key differentiation genes, many of which are mutated in human skin diseases, including FLG, LOR, ALOXE3, ALOX12B, ABCA12, CASP14 and ELOVL3. TINCR-deficient epidermis lacked terminal differentiation ultrastructure, including keratohyalin granules and intact lamellar bodies. Genome-scale RNA interactome analysis revealed that TINCR interacts with a range of differentiation mRNAs. TINCR-mRNA interaction occurs through a 25-nucleotide 'TINCR box' motif that is strongly enriched in interacting mRNAs and required for TINCR binding. A high-throughput screen to analyse TINCR binding capacity to approximately 9,400 human recombinant proteins revealed direct binding of TINCR RNA to the staufen1 (STAU1) protein. STAU1-deficient tissue recapitulated the impaired differentiation seen with TINCR depletion. Loss of UPF1 and UPF2, both of which are required for STAU1-mediated RNA decay, however, did not have differentiation effects. Instead, the TINCR-STAU1 complex seems to mediate stabilization of differentiation mRNAs, such as KRT80. These data identify TINCR as a key lncRNA required for somatic tissue differentiation, which occurs through lncRNA binding to differentiation mRNAs to ensure their expression.

Identification of proteins binding coding and non-coding human RNAs using protein microarrays.

BACKGROUND: The regulation and function of mammalian RNAs has been increasingly appreciated to operate via RNA-protein interactions. With the recent discovery of thousands of novel human RNA molecules by high-throughput RNA sequencing, efficient methods to uncover RNA-protein interactions are urgently required. Existing methods to study proteins associated with a given RNA are laborious and require substantial amounts of cell-derived starting material. To overcome these limitations, we have developed a rapid and large-scale approach to characterize binding of in vitro transcribed labeled RNA to ~9,400 human recombinant proteins spotted on protein microarrays. RESULTS: We have optimized methodology to probe human protein microarrays with full-length RNA molecules and have identified 137 RNA-protein interactions specific for 10 coding and non-coding RNAs. Those proteins showed strong enrichment for common human RNA binding domains such as RRM, RBD, as well as K homology and CCCH type zinc finger motifs. Previously unknown RNA-protein interactions were discovered using this technique, and these interactions were biochemically verified between TP53 mRNA and Staufen1 protein as well as between HRAS mRNA and CNBP protein. Functional characterization of the interaction between Staufen 1 protein and TP53 mRNA revealed a novel role for Staufen 1 in preserving TP53 RNA stability. CONCLUSIONS: Our approach demonstrates a scalable methodology, allowing rapid and efficient identification of novel human RNA-protein interactions using RNA hybridization to human protein microarrays. Biochemical validation of newly identified interactions between TP53-Stau1 and HRAS-CNBP using reciprocal pull-down experiments, both in vitro and in vivo, demonstrates the utility of this approach to study uncharacterized RNA-protein interactions.

Transcriptome sequencing in Sezary syndrome identifies Sezary cell and mycosis fungoides-associated lncRNAs and novel transcripts.

Sézary syndrome (SS) is an aggressive cutaneous T-cell lymphoma (CTCL) of unknown etiology in which malignant cells circulate in the peripheral blood. To identify viral elements, gene fusions, and gene expression patterns associated with this lymphoma, flow cytometry was used to obtain matched pure populations of malignant Sézary cells (SCs) versus nonmalignant CD4(+) T cells from 3 patients for whole transcriptome, paired-end sequencing with an average depth of 112 million reads per sample. Pathway analysis of differentially expressed genes identified mis-regulation of PI3K/Akt, TGFß, and NF-κB pathways as well as T-cell receptor signaling. Bioinformatic analysis did not detect either nonhuman transcripts to support a viral etiology of SS or recurrently expressed gene fusions, but it did identify 21 SC-associated annotated long noncoding RNAs (lncRNAs). Transcriptome assembly by multiple algorithms identified 13 differentially expressed unannotated transcripts termed Sézary cell-associated transcripts (SeCATs) that include 12 predicted lncRNAs and a novel transcript with coding potential. High-throughput sequencing targeting the 3' end of polyadenylated transcripts in archived tumors from 24 additional patients with tumor-stage CTCL confirmed the differential expression of SC-associated lncRNAs and SeCATs in CTCL. Our findings characterize the SS transcriptome and support recent reports that implicate lncRNA dysregulation in human malignancies.

BRAFV600E remodels the melanocyte transcriptome and induces BANCR to regulate melanoma cell migration.

Aberrations of protein-coding genes are a focus of cancer genomics; however, the impact of oncogenes on expression of the ~50% of transcripts without protein-coding potential, including long noncoding RNAs (lncRNAs), has been largely uncharacterized. Activating mutations in the BRAF oncogene are present in >70% of melanomas, 90% of which produce active mutant BRAF(V600E) protein. To define the impacts of oncogenic BRAF on the melanocyte transcriptome, massively parallel cDNA sequencing (RNA-seq) was performed on genetically matched normal human melanocytes with and without BRAF(V600E) expression. To enhance potential disease relevance by verifying expression of altered genes in BRAF-driven cancer tissue, parallel RNA-seq was also undertaken of two BRAF(V600E)-mutant human melanomas. BRAF(V600E) regulated expression of 1027 protein-coding transcripts and 39 annotated lncRNAs, as well as 70 unannotated, potentially novel, intergenic transcripts. These transcripts display both tissue-specific and multi-tissue expression profiles and harbor distinctive regulatory chromatin marks and transcription factor binding sites indicative of active transcription. Coding potential analysis of the 70 unannotated transcripts suggested that most may represent newly identified lncRNAs. BRAF-regulated lncRNA 1 (BANCR) was identified as a recurrently overexpressed, previously unannotated 693-bp transcript on chromosome 9 with a potential functional role in melanoma cell migration. BANCR knockdown reduced melanoma cell migration, and this could be rescued by the chemokine CXCL11. Combining RNA-seq of oncogene-expressing normal cells with RNA-seq of their corresponding human cancers may represent a useful approach to discover new oncogene-regulated RNA transcripts of potential clinical relevance in cancer.