TERRA RNA Antagonizes ATRX and Protects Telomeres.

Through an integration of genomic and proteomic approaches to advance understanding of long noncoding RNAs, we investigate the function of the telomeric transcript, TERRA. By identifying thousands of TERRA target sites in the mouse genome, we demonstrate that TERRA can bind both in cis to telomeres and in trans to genic targets. We then define a large network of interacting proteins, including epigenetic factors, telomeric proteins, and the RNA helicase, ATRX. TERRA and ATRX share hundreds of target genes and are functionally antagonistic at these loci: whereas TERRA activates, ATRX represses gene expression. At telomeres, TERRA competes with telomeric DNA for ATRX binding, suppresses ATRX localization, and ensures telomeric stability. Depleting TERRA increases telomerase activity and induces telomeric pathologies, including formation of telomere-induced DNA damage foci and loss or duplication of telomeric sequences. We conclude that TERRA functions as an epigenomic modulator in trans and as an essential regulator of telomeres in cis.

Destabilization of B2 RNA by EZH2 Activates the Stress Response.

More than 98% of the mammalian genome is noncoding, and interspersed transposable elements account for ∼50% of noncoding space. Here, we demonstrate that a specific interaction between the polycomb protein EZH2 and RNA made from B2 SINE retrotransposons controls stress-responsive genes in mouse cells. In the heat-shock model, B2 RNA binds stress genes and suppresses their transcription. Upon stress, EZH2 is recruited and triggers cleavage of B2 RNA. B2 degradation in turn upregulates stress genes. Evidence indicates that B2 RNA operates as a "speed bump" against advancement of RNA polymerase II, and temperature stress releases the brakes on transcriptional elongation. These data attribute a new function to EZH2 that is independent of its histone methyltransferase activity and reconcile how EZH2 can be associated with both gene repression and activation. Our study reveals that EZH2 and B2 together control activation of a large network of genes involved in thermal stress.

ATRX directs binding of PRC2 to Xist RNA and Polycomb targets.

X chromosome inactivation (XCI) depends on the long noncoding RNA Xist and its recruitment of Polycomb Repressive Complex 2 (PRC2). PRC2 is also targeted to other sites throughout the genome to effect transcriptional repression. Using XCI as a model, we apply an unbiased proteomics approach to isolate Xist and PRC2 regulators and identified ATRX. ATRX unexpectedly functions as a high-affinity RNA-binding protein that directly interacts with RepA/Xist RNA to promote loading of PRC2 in vivo. Without ATRX, PRC2 cannot load onto Xist RNA nor spread in cis along the X chromosome. Moreover, epigenomic profiling reveals that genome-wide targeting of PRC2 depends on ATRX, as loss of ATRX leads to spatial redistribution of PRC2 and derepression of Polycomb responsive genes. Thus, ATRX is a required specificity determinant for PRC2 targeting and function.

A disproportionate impact of G9a methyltransferase deficiency on the X chromosome.

G9a is a histone methyltransferase responsible for the dimethylation of histone H3 at lysine 9 (H3K9me2). G9a plays key roles in transcriptional silencing of developmentally regulated genes, but its role in X-chromosome inactivation (XCI) has been under debate. Here, we uncover a female-specific function of G9a and demonstrate that deleting G9a has a disproportionate impact on the X chromosome relative to the rest of the genome. G9a deficiency causes a failure of XCI and female-specific hypersensitivity to drug inhibition of H3K9me2. We show that G9a interacts with Tsix and Xist RNAs, and that competitive inhibition of the G9a-RNA interaction recapitulates the XCI defect. During XCI, Xist recruits G9a to silence X-linked genes on the future inactive X. In parallel on the future Xa, Tsix recruits G9a to silence Xist in cis Thus, RNA tethers G9a for allele-specific targeting of the H3K9me2 modification and the G9a-RNA interaction is essential for XCI.

Toward a consensus on the binding specificity and promiscuity of PRC2 for RNA.

Polycomb repressive complex-2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. Early works suggested binding specificity of PRC2 to certain long non-coding RNAs for recruitment to chromatin. More recent studies provided evidence both in favor and against this idea. Here, we bridge the two existing models of PRC2-RNA interaction. RepA RNA is a good binding partner for PRC2, while multiple non-relevant RNAs, including bacterial mRNAs, also bind PRC2; Kds depend to some extent on the experimental conditions. Human and mouse PRC2 have broadly similar RNA-binding properties in vitro. Examination of evidence supporting an existing model for site-specific recruitment of PRC2 by a well-defined RNA motif in cells reveals that results are PRC2 independent. We conclude that promiscuous and specific RNA-binding activities of PRC2 in vitro are not mutually exclusive, and that binding specificity in vivo remains to be demonstrated.

Locus-specific targeting to the X chromosome revealed by the RNA interactome of CTCF.

CTCF is a master regulator that plays important roles in genome architecture and gene expression. How CTCF is recruited in a locus-specific manner is not fully understood. Evidence from epigenetic processes, such as X chromosome inactivation (XCI), indicates that CTCF associates functionally with RNA. Using genome-wide approaches to investigate the relationship between its RNA interactome and epigenomic landscape, here we report that CTCF binds thousands of transcripts in mouse embryonic stem cells, many in close proximity to CTCF's genomic binding sites. CTCF is a specific and high-affinity RNA-binding protein (Kd < 1 nM). During XCI, CTCF differentially binds the active and inactive X chromosomes and interacts directly with Tsix, Xite, and Xist RNAs. Tsix and Xite RNAs target CTCF to the X inactivation center, thereby inducing homologous X chromosome pairing. Our work elucidates one mechanism by which CTCF is recruited in a locus-specific manner and implicates CTCF-RNA interactions in long-range chromosomal interactions.

B2 and ALU retrotransposons are self-cleaving ribozymes whose activity is enhanced by EZH2.

Transposable elements make up half of the mammalian genome. One of the most abundant is the short interspersed nuclear element (SINE). Among their million copies, B2 accounts for ∼350,000 in the mouse genome and has garnered special interest because of emerging roles in epigenetic regulation. Our recent work demonstrated that B2 RNA binds stress genes to retard transcription elongation. Although epigenetically silenced, B2s become massively up-regulated during thermal and other types of stress. Specifically, an interaction between B2 RNA and the Polycomb protein, EZH2, results in cleavage of B2 RNA, release of B2 RNA from chromatin, and activation of thermal stress genes. Although an established RNA-binding protein and histone methyltransferase, EZH2 is not known to be a nuclease. Here, we provide evidence for the surprising conclusion that B2 is a self-cleaving ribozyme. Ribozyme activity depends on Mg+2 and monovalent cations but is resistant to protease treatment. However, contact with EZH2 accelerates cleavage rate by >100-fold, suggesting that EZH2 promotes a cleavage-competent RNA conformation. B2 modification-interference analysis demonstrates that phosphorothioate changes at A and C nucleotides can substitute for EZH2. B2 nucleotides 45 to 55 and 100 to 101 are essential for activity. Finally, another family of SINEs, the human ALU element, also produces a self-cleaving RNA and is cleaved during T-cell activation as well as thermal and endoplasmic reticulum (ER) stress. Thus, B2/ALU SINEs may be classified as "epigenetic ribozymes" that function as transcriptional switches during stress. Given their high copy numbers, B2 and ALU may represent the predominant ribozyme activity in mammalian cells.

Regulatory interactions between RNA and polycomb repressive complex 2.

Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that is localized to thousands of mammalian genes. Though important to human disease and as a drug target, how PRC2 is recruited remains unclear. One model invokes cis-regulatory RNA. Herein, we biochemically and functionally probe PRC2's recognition of RNA using the X-inactivation model. We observe surprisingly high discriminatory capabilities. While SUZ12 and JARID2 subunits can bind RNA, EZH2 has highest affinity and is somewhat promiscuous. EED regulates the affinity of EZH2 for RNA, lending greater specificity to PRC2-RNA interactions. Intriguingly, while RNA is crucial for targeting, RNA inhibits EZH2's catalytic activity. JARID2 weakens PRC2's binding to RNA and relieves catalytic inhibition. We propose that RNA guides PRC2 to its target but inhibits its enzymatic activity until PRC2 associates with JARID2 on chromatin. Our study provides a molecular view of regulatory interactions between RNA and PRC2 at the chromatin interface.

An alternative telomerase RNA in Arabidopsis modulates enzyme activity in response to DNA damage.

Telomerase replenishes telomere tracts by reiteratively copying its RNA template, TER. Unlike other model organisms, Arabidopsis thaliana harbors two divergent TER genes. However, only TER1 is required for telomere maintenance. Here we examine the function of TER2. We show that TER2 is spliced and its 3' end is truncated in vivo to generate a third TER isoform, TER2(S). TERT preferentially associates with TER2 > TER1 > TER2(S). Moreover, TER2 and TER2(S) assemble with Ku and POT1b (protection of telomeres), forming RNP (ribonucleoprotein) complexes distinct from TER1 RNP. Plants null for TER2 display increased telomerase enzyme activity, while TER2 overexpression inhibits telomere synthesis from TER1 and leads to telomere shortening. These findings argue that TER2 negatively regulates telomerase by sequestering TERT in a nonproductive RNP complex. Introduction of DNA double-strand breaks by zeocin leads to an immediate and specific spike in TER2 and a concomitant decrease in telomerase enzyme activity. This response is not triggered by replication stress or telomere dysfunction and is abrogated in ter2 mutants. We conclude that Arabidopsis telomerase is modulated by TER2, a novel DNA damage-induced noncoding RNA that works in concert with the canonical TER to promote genome integrity.

Functional Proteomic Analysis of Repressive Histone Methyltransferase Complexes Reveals ZNF518B as a G9A Regulator.

Cell-type specific gene silencing by histone H3 lysine 27 and lysine 9 methyltransferase complexes PRC2 and G9A-GLP is crucial both during development and to maintain cell identity. Although studying their interaction partners has yielded valuable insight into their functions, how these factors are regulated on a network level remains incompletely understood. Here, we present a new approach that combines quantitative interaction proteomics with global chromatin profiling to functionally characterize repressive chromatin modifying protein complexes in embryonic stem cells. We define binding stoichiometries of 9 new and 12 known interaction partners of PRC2 and 10 known and 29 new interaction partners of G9A-GLP, respectively. We demonstrate that PRC2 and G9A-GLP interact physically and share several interaction partners, including the zinc finger proteins ZNF518A and ZNF518B. Using global chromatin profiling by targeted mass spectrometry, we discover that even sub-stoichiometric binding partners such as ZNF518B can positively regulate global H3K9me2 levels. Biochemical analysis reveals that ZNF518B directly interacts with EZH2 and G9A. Our systematic analysis suggests that ZNF518B may mediate the structural association between PRC2 and G9A-GLP histone methyltransferases and additionally regulates the activity of G9A-GLP.

Two RNA subunits and POT1a are components of Arabidopsis telomerase.

Telomerase is a ribonucleoprotein (RNP) reverse transcriptase whose essential RNA subunit (TER) functions as a template for telomere repeat synthesis. Here we report the identification of two divergent TER moieties in the flowering plant Arabidopsis thaliana. Although both TER1 and TER2 copurify with telomerase activity and serve as templates for telomerase in vitro, depletion of TER1, but not TER2, leads to decreased telomerase activity and progressive telomere shortening in vivo. Moreover, mutation of the templating domain in TER1 results in the incorporation of mutant telomere repeats on chromosome ends. Thus, TER1 provides the major template for telomerase in vivo. We also show that POT1a binds TER1 with a Kd of 2 × 10(-7) M and the two components assemble into an enzymatically active RNP in vivo. In contrast, TER1-POT1b and TER2-POT1a associations were not observed. In other organisms POT1 proteins bind telomeric DNA and provide chromosome end protection. We propose that duplication of TER and POT1 in Arabidopsis fueled the evolution of novel protein-nucleic acid interactions and the migration of POT1 from the telomere to the telomerase RNP.

Telomerase regulation.

The intimate connection between telomerase regulation and human disease is now well established. The molecular basis for telomerase regulation is highly complex and entails multiple layers of control. While the major target of enzyme regulation is the catalytic subunit TERT, the RNA subunit of telomerase is also implicated in telomerase control. In addition, alterations in gene dosage and alternative isoforms of core telomerase components have been described. Finally, telomerase localization, recruitment to the telomere and enzymology at the chromosome terminus are all subject to modulation. In this review we summarize recent advances in understanding fundamental mechanisms of telomerase regulation.

Minimal pre-mRNA substrates with natural and converted sites for full-round U insertion and U deletion RNA editing in trypanosomes.

Trypanosome RNA editing by uridylate insertion or deletion cycles is a mitochondrial mRNA maturation process catalyzed by multisubunit complexes. A full-round of editing entails three consecutive steps directed by partially complementary guide RNAs: pre-mRNA cleavage, U addition or removal, and ligation. The structural and functional composition of editing complexes is intensively studied, but their molecular interactions in and around editing sites are not completely understood. In this study, we performed a systematic analysis of distal RNA requirements for full-round insertion and deletion by purified editosomes. We define minimal substrates for efficient editing of A6 and CYb model transcripts, and established a new substrate, RPS12. Important differences were observed in the composition of substrates for insertion and deletion. Furthermore, we also showed for the first time that natural sites can be artificially converted in both directions: from deletion to insertion or from insertion to deletion. Our site conversions enabled a direct comparison of the two editing kinds at common sites during substrate minimization and demonstrate that all basic determinants directing the editosome to carry out full-round insertion or deletion reside within each editing site. Surprisingly, we were able to engineer a deletion site into CYb, which exclusively undergoes insertion in nature.

Determinants for association and guide RNA-directed endonuclease cleavage by purified RNA editing complexes from Trypanosoma brucei.

U-insertion/deletion RNA editing in the single mitochondrion of kinetoplastids, an ancient lineage of eukaryotes, is a unique mRNA maturation process needed for translation. Multisubunit editing complexes recognize many pre-edited mRNA sites and modify them via cycles of three catalytic steps: guide RNA (gRNA)-directed cleavage, insertion or deletion of uridylates at the 3'-terminus of the upstream cleaved piece, and ligation of the two mRNA pieces. While catalytic and many structural protein subunits of these complexes have been identified, the mechanisms and basic determinants of substrate recognition are still poorly understood. This study defined relatively simple single- and double-stranded determinants for association and gRNA-directed cleavage. To this end, we used an electrophoretic mobility shift assay to directly score the association of purified editing complexes with RNA ligands, in parallel with UV photocrosslinking and functional studies. The cleaved strand required a minimal 5' overhang of 12 nt and an approximately 15-bp duplex with gRNA to direct the cleavage site. A second protruding element in either the cleaved or the guide strand was required unless longer duplexes were used. Importantly, the single-stranded RNA requirement for association can be upstream or downstream of the duplex, and the binding and cleavage activities of purified editing complexes could be uncoupled. The current observations together with our previous reports in the context of purified native editing complexes show that the determinants for association, cleavage and full-round editing gradually increase in complexity as these stages progress. The native complexes in these studies contained most, if not all, known core subunits in addition to components of the MRP complex. Finally, we found that the endonuclease KREN1 in purified complexes photocrosslinks with a targeted editing site. A model is proposed whereby one or more RNase III-type endonucleases mediate the initial binding and scrutiny of potential ligands and subsequent catalytic selectivity triggers either insertion or deletion editing enzymes.

Substrate determinants for RNA editing and editing complex interactions at a site for full-round U insertion.

Multisubunit RNA editing complexes catalyze uridylate insertion/deletion RNA editing directed by complementary guide RNAs (gRNAs). Editing in trypanosome mitochondria is transcript-specific and developmentally controlled, but the molecular mechanisms of substrate specificity remain unknown. Here we used a minimal A6 pre-mRNA/gRNA substrate to define functional determinants for full-round insertion and editing complex interactions at the editing site 2 (ES2). Editing begins with pre-mRNA cleavage within an internal loop flanked by upstream and downstream duplexes with gRNA. We found that substrate recognition around the internal loop is sequence-independent and that completely artificial duplexes spanning a single helical turn are functional. Furthermore, after our report of cross-linking interactions at the deletion ES1 (35), we show for the first time editing complex contacts at an insertion ES. Our studies using site-specific ribose 2' substitutions defined 2'-hydroxyls within the (a) gRNA loop region and (b) flanking helixes that markedly stimulate both pre-mRNA cleavage and editing complex interactions at ES2. Modification of the downstream helix affected scissile bond specificity. Notably, a single 2'-hydroxyl at ES2 is essential for cleavage but dispensable for editing complex cross-linking. This study provides new insights on substrate recognition during full-round editing, including the relevance of secondary structure and the first functional association of specific (pre-mRNA and gRNA) riboses with both endonuclease cleavage and cross-linking activities of editing complexes at an ES. Importantly, most observed cross-linking interactions are both conserved and relatively stable at ES2 and ES1 in hybrid substrates. However, they were also detected as transient low-stability contacts in a non-edited transcript.

RNA editing complex interactions with a site for full-round U deletion in Trypanosoma brucei.

Trypanosome U insertion and U deletion RNA editing of mitochondrial pre-mRNAs is catalyzed by multisubunit editing complexes as directed by partially complementary guide RNAs. The basic enzymatic activities and protein composition of these high-molecular mass complexes have been under intense study, but their specific protein interactions with functional pre-mRNA/gRNA substrates remains unknown. We show that editing complexes purified through extensive ion-exchange chromatography and immunoprecipitation make specific cross-linking interactions with A6 pre-mRNA containing a single 32P and photoreactive 4-thioU at the scissile bond of a functional site for full-round U deletion. At least four direct protein-RNA contacts are detected at this site by cross-linking. All four interactions are stimulated by unpaired residues just 5' of the pre-mRNA/gRNA anchor duplex, but strongly inhibited by pairing of the editing site region. Furthermore, competition analysis with homologous and heterologous transcripts suggests preferential contacts of the editing complex with the mRNA/gRNA duplex substrate. This apparent structural selectivity suggests that the RNA-protein interactions we observe may be involved in recognition of editing sites and/or catalysis in assembled complexes.

Valores de referencia de células asesinas naturales (NK y NKT) en donantes de sangre de Bogotá / Reference values of the natural killer cells (NK and NKT) on blood donors in Bogota

Introducción: las células asesinas naturales (NK y NKT) son una población de linfocitos que circulan en bajos porcentajes en sangre periférica. Ambos tipos de células realizan un papel importante en la respuesta inmune en condiciones normales o en procesos patológicos como ciertos tumores,abortos espontáneos y en el rechazo a trasplantes, lo cual les confiere un gran interés clínico. Objetivo: el presente trabajo pretende establecer valores para las células NK y NKT por medio decitometría de flujo en una población adulta de donantes de un banco de sangre de Bogotá. Metodología: se recolectaron 104 muestras de donantes y se realizó un estudio con los marcadores CD3, CD16 y CD56.Resultados: de las muestras 47.2% fueron mujeres y 52.8% hombres. Para las mujeres el porcentajede células fue: NK 14.6% (μ 12.1) y NKT 3.0% (μ 2.5); para hombres NK 25.3% (μ 21.3) y NKT 3.5% (μ 2.9). Los valores absolutos de células para mujeres fueron NK 298.8 /µL (μ 253.9) y NKT 70.1 /µL (μ 47.7); para hombres NK 526.1 /µL (μ 448) y NKT 95.5 /µL (μ 77). Existe una diferencia estadística entre los valores absolutos par las células NK entre hombres y mujeres (p= 0.0004).

Introduction: the natural killer cells (NK y NKT) are a population of lymphocytes that circulate in the peripheral blood but in low percentages. Both types of cells play an important role in immuneresponse under normal conditions or in pathological processes as it is the case of certain tumors, spontaneous abortions and transplant rejections, what makes them very interesting from the clinical interest point of view.Objective: the aim of this work is to establish values for NK and NKT cells by means of flow cytometry in a group of adult population of blood donors in a blood bank in Bogotá. Methodology: 104 donor samples were collected and a study was carried out with CD3, CD56and CD16 markers. Results: 47.2% of the samples were women and 52.8% men. In the case of women the cellspercentage was: NK 14.6% (μ 12.1) and NKT 3.0% (μ 2.5); for men NK 25.3% (μ 21.3) and NKT 3.5% (μ 2.9). The absolute cells values for women were NK 298.8 /µL (μ 253.9) and NKT 70.1 /µL (μ 47.7); for men NK 526.1 /µL (μ 448) and NKT 95.5 /µL (μ 77). There is a statistical differencebetween the absolute values by the NK cells between men and women. (p= 0.0004).