The Long Non-Coding RNA ANRIL in Cancers.

ANRIL (Antisense Noncoding RNA in the INK4 Locus), a long non-coding RNA encoded in the human chromosome 9p21 region, is a critical factor for regulating gene expression by interacting with multiple proteins and miRNAs. It has been found to play important roles in various cellular processes, including cell cycle control and proliferation. Dysregulation of ANRIL has been associated with several diseases like cancers and cardiovascular diseases, for instance. Understanding the oncogenic role of ANRIL and its potential as a diagnostic and prognostic biomarker in cancer is crucial. This review provides insights into the regulatory mechanisms and oncogenic significance of the 9p21 locus and ANRIL in cancer.

Emerging role of IκBζ in inflammation: Emphasis on psoriasis.

Psoriasis is a chronic inflammatory disorder affecting skin and joints that results from immunological dysfunction such as enhanced IL-23 induced Th-17 differentiation. IkappaB-Zeta (IκBζ) is an atypical transcriptional factor of the IκB protein family since, contrary to the other family members, it positively regulates NF-κB pathway by being exclusively localized into the nucleus. IκBζ deficiency reduces visible manifestations of experimental psoriasis by diminishing expression of psoriasis-associated genes. It is thus tempting to consider IκBζ as a potential therapeutic target for psoriasis as well as for other IL23/IL17-mediated inflammatory diseases. In this review, we will discuss the regulation of expression of NFKBIZ and its protein IκBζ, its downstream targets, its involvement in pathogenesis of multiple disorders with emphasis on psoriasis and evidences supporting that inhibition of IκBζ may be a promising alternative to current therapeutic managements of psoriasis.

Implication of repeat insertion domains in the trans-activity of the long non-coding RNA ANRIL.

Long non-coding RNAs have emerged as critical regulators of cell homeostasis by modulating gene expression at chromatin level for instance. Here, we report that the lncRNA ANRIL, associated with several pathologies, binds to thousands of loci dispersed throughout the mammalian genome sharing a 21-bp motif enriched in G/A residues. By combining ANRIL genomic occupancy with transcriptomic analysis, we established a list of 65 and 123 genes potentially directly activated and silenced by ANRIL in trans, respectively. We also found that Exon8 of ANRIL, mainly made of transposable elements, contributes to ANRIL genomic association and consequently to its trans-activity. Furthermore, we showed that Exon8 favors ANRIL's association with the FIRRE, TPD52L1 and IGFBP3 loci to modulate their expression through H3K27me3 deposition. We also investigated the mechanisms engaged by Exon8 to favor ANRIL's association with the genome. Our data refine ANRIL's trans-activity and highlight the functional importance of TEs on ANRIL's activity.

Non-Coding RNA Silencing in Mammalian Cells by Antisense LNA GapmeRs Transfection.

The assessment of non-coding RNAs (ncRNAs) functions highly relies on loss of function studies. However, due to their exclusive or partial nuclear localization, many small and long ncRNAs are not efficiently silenced by RNA interference. Antisense LNA GapmeRs constitute a good alternative to RNAi. They allow an effective knockdown of ncRNAs with sizes greater than 80 nucleotides, regardless of their cellular localization. This chapter focuses on the silencing of two different nuclear ncRNAs (ANRIL and SATIII RNAs) in mammalian cells using antisense LNA GapmeRs with two different transfection methods: calcium phosphate-mediated transfection and LipofectamineTM 2000.

Purification of RiboNucleoProtein Particles by MS2-MBP Affinity Chromatography.

RiboNucleoProtein particles (RNPs), which are composed of RNAs and proteins, play essential roles in many biological processes. The isolation of these molecular machines is a critical step to better understand their mechanisms of action. In this chapter, we describe the MS2-MBP affinity chromatography used to purify the protein content of the RNPs formed with an RNA of interest in a nuclear extract. Substrate RNAs are furnished with a tag consisting of three stem-loops that provide specific binding sites for the phage MS2 protein. Here, we successfully applied this method to isolate RNPs formed with subfragments of the long noncoding RNA ANRIL (Antisense Noncoding RNA in the INK4 Locus).

Study of Genome-Wide Occupancy of Long Non-Coding RNAs Using Chromatin Isolation by RNA Purification (ChIRP).

Long noncoding RNAs (lncRNAs) have recently emerged as masters of gene expression regulation by exerting their functions in all cell compartments through a wide repertoire of mechanisms. A high portion of lncRNAs are robustly enriched in the chromatin fraction suggesting a broad regulatory role in the nuclear compartment. Despite the advances in this field, the interaction between lncRNAs and the chromatin is still poorly understood. This led to the emergence of numerous hybridization capture assays such as the Chromatin Isolation by RNA Purification (ChIRP) which revealed at high resolution the genomic binding sites of several nuclear lncRNAs. In this chapter, we describe the ChIRP protocol that was successfully applied to the lncRNA ANRIL. We also provide a user-friendly bioinformatic pipeline for ChIRP-seq data analysis.

JASPer controls interphase histone H3S10 phosphorylation by chromosomal kinase JIL-1 in Drosophila.

In flies, the chromosomal kinase JIL-1 is responsible for most interphase histone H3S10 phosphorylation and has been proposed to protect active chromatin from acquiring heterochromatic marks, such as dimethylated histone H3K9 (H3K9me2) and HP1. Here, we show that JIL-1's targeting to chromatin depends on a PWWP domain-containing protein JASPer (JIL-1 Anchoring and Stabilizing Protein). JASPer-JIL-1 (JJ)-complex is the major form of kinase in vivo and is targeted to active genes and telomeric transposons via binding of the PWWP domain of JASPer to H3K36me3 nucleosomes, to modulate transcriptional output. JIL-1 and JJ-complex depletion in cycling cells lead to small changes in H3K9me2 distribution at active genes and telomeric transposons. Finally, we identify interactors of the endogenous JJ-complex and propose that JIL-1 not only prevents heterochromatin formation but also coordinates chromatin-based regulation in the transcribed part of the genome.

Live cell imaging of the nascent inactive X chromosome during the early differentiation process of naive ES cells towards epiblast stem cells.

Random X-chromosome inactivation ensures dosage compensation in mammals through the transcriptional silencing of one of the two X chromosomes present in each female cell. Silencing is initiated in the differentiating epiblast of the mouse female embryos through coating of the nascent inactive X chromosome by the non-coding RNA Xist, which subsequently recruits the Polycomb Complex PRC2 leading to histone H3-K27 methylation. Here we examined in mouse ES cells the early steps of the transition from naive ES cells towards epiblast stem cells as a model for inducing X chromosome inactivation in vitro. We show that these conditions efficiently induce random XCI. Importantly, in a transient phase of this differentiation pathway, both X chromosomes are coated with Xist RNA in up to 15% of the XX cells. In an attempt to determine the dynamics of this process, we designed a strategy aimed at visualizing the nascent inactive X-chromosome in live cells. We generated transgenic female XX ES cells expressing the PRC2 component Ezh2 fused to the fluorescent protein Venus. The fluorescent fusion protein was expressed at sub-physiological levels and located in nuclei of ES cells. Upon differentiation of ES cell towards epiblast stem cell fate, Venus-fluorescent territories appearing in interphase nuclei were identified as nascent inactive X chromosomes by their association with Xist RNA. Imaging of Ezh2-Venus for up to 24 hours during the differentiation process showed survival of some cells with two fluorescent domains and a surprising dynamics of the fluorescent territories across cell division and in the course of the differentiation process. Our data reveal a strategy for visualizing the nascent inactive X chromosome and suggests the possibility for a large plasticity of the nascent inactive X chromosome.

UNR facilitates the interaction of MLE with the lncRNA roX2 during Drosophila dosage compensation.

Dosage compensation is a regulatory process that balances the expression of X-chromosomal genes between males (XY) and females (XX). In Drosophila, this requires non-coding RNAs and RNA-binding proteins (RBPs) whose specific functions remain elusive. Here we show that the Drosophila RBP UNR promotes the targeting of the activating male-specific-lethal complex to the X-chromosome by facilitating the interaction of two crucial subunits: the RNA helicase MLE and the long non-coding RNA roX2.

ATP-dependent roX RNA remodeling by the helicase maleless enables specific association of MSL proteins.

Dosage compensation in Drosophila involves a global activation of genes on the male X chromosome. The activating complex (MSL-DCC) consists of male-specific-lethal (MSL) proteins and two long, noncoding roX RNAs. The roX RNAs are essential for X-chromosomal targeting, but their contributions to MSL-DCC structure and function are enigmatic. Conceivably, the RNA helicase MLE, itself an MSL subunit, is actively involved in incorporating roX into functional DCC. We determined the secondary structure of roX2 and mapped specific interaction sites for MLE in vitro. Upon addition of ATP, MLE disrupted a functionally important stem loop in roX2. This RNA remodeling enhanced specific ATP-dependent association of MSL2, the core subunit of the MSL-DCC, providing a link between roX and MSL subunits. Probing the conformation of roX in vivo revealed a remodeled stem loop in chromatin-bound roX2. The active remodeling of a stable secondary structure by MLE may constitute a rate-limiting step for MSL-DCC assembly.

Roles of long, non-coding RNA in chromosome-wide transcription regulation: lessons from two dosage compensation systems.

A large part of higher eukaryotic genomes is transcribed into RNAs lacking any significant open reading frame. This "non-coding part" has been shown to actively contribute to regulating gene expression, but the mechanisms are largely unknown. Particularly instructive examples are provided by the dosage compensation systems, which assure that the single X chromosome in male cells and the two X chromosomes in female cells give rise to similar amounts of gene product. Although this is achieved by very different strategies in mammals and fruit flies, long, non-coding RNAs (lncRNAs) are involved in both cases. Here we summarize recent progress towards unraveling the mechanisms, by which the Xist and roX RNAs mediate the selective association of regulators with individual target chromosomes, to initiate dosage compensation in mammals and fruit flies, respectively.

2-D structure of the A region of Xist RNA and its implication for PRC2 association.

In placental mammals, inactivation of one of the X chromosomes in female cells ensures sex chromosome dosage compensation. The 17 kb non-coding Xist RNA is crucial to this process and accumulates on the future inactive X chromosome. The most conserved Xist RNA region, the A region, contains eight or nine repeats separated by U-rich spacers. It is implicated in the recruitment of late inactivated X genes to the silencing compartment and likely in the recruitment of complex PRC2. Little is known about the structure of the A region and more generally about Xist RNA structure. Knowledge of its structure is restricted to an NMR study of a single A repeat element. Our study is the first experimental analysis of the structure of the entire A region in solution. By the use of chemical and enzymatic probes and FRET experiments, using oligonucleotides carrying fluorescent dyes, we resolved problems linked to sequence redundancies and established a 2-D structure for the A region that contains two long stem-loop structures each including four repeats. Interactions formed between repeats and between repeats and spacers stabilize these structures. Conservation of the spacer terminal sequences allows formation of such structures in all sequenced Xist RNAs. By combination of RNP affinity chromatography, immunoprecipitation assays, mass spectrometry, and Western blot analysis, we demonstrate that the A region can associate with components of the PRC2 complex in mouse ES cell nuclear extracts. Whilst a single four-repeat motif is able to associate with components of this complex, recruitment of Suz12 is clearly more efficient when the entire A region is present. Our data with their emphasis on the importance of inter-repeat pairing change fundamentally our conception of the 2-D structure of the A region of Xist RNA and support its possible implication in recruitment of the PRC2 complex.