Long noncoding RNAs with enhancer-like function in human cells.

While the long noncoding RNAs (ncRNAs) constitute a large portion of the mammalian transcriptome, their biological functions has remained elusive. A few long ncRNAs that have been studied in any detail silence gene expression in processes such as X-inactivation and imprinting. We used a GENCODE annotation of the human genome to characterize over a thousand long ncRNAs that are expressed in multiple cell lines. Unexpectedly, we found an enhancer-like function for a set of these long ncRNAs in human cell lines. Depletion of a number of ncRNAs led to decreased expression of their neighboring protein-coding genes, including the master regulator of hematopoiesis, SCL (also called TAL1), Snai1 and Snai2. Using heterologous transcription assays we demonstrated a requirement for the ncRNAs in activation of gene expression. These results reveal an unanticipated role for a class of long ncRNAs in activation of critical regulators of development and differentiation.

EPOP Functionally Links Elongin and Polycomb in Pluripotent Stem Cells.

The cellular plasticity of pluripotent stem cells is thought to be sustained by genomic regions that display both active and repressive chromatin properties. These regions exhibit low levels of gene expression, yet the mechanisms controlling these levels remain unknown. Here, we describe Elongin BC as a binding factor at the promoters of bivalent sites. Biochemical and genome-wide analyses show that Elongin BC is associated with Polycomb Repressive Complex 2 (PRC2) in pluripotent stem cells. Elongin BC is recruited to chromatin by the PRC2-associated factor EPOP (Elongin BC and Polycomb Repressive Complex 2 Associated Protein, also termed C17orf96, esPRC2p48, E130012A19Rik), a protein expressed in the inner cell mass of the mouse blastocyst. Both EPOP and Elongin BC are required to maintain low levels of expression at PRC2 genomic targets. Our results indicate that keeping the balance between activating and repressive cues is a more general feature of chromatin in pluripotent stem cells than previously appreciated.

Differential contribution to gene expression prediction of histone modifications at enhancers or promoters.

The ChIP-seq signal of histone modifications at promoters is a good predictor of gene expression in different cellular contexts, but whether this is also true at enhancers is not clear. To address this issue, we develop quantitative models to characterize the relationship of gene expression with histone modifications at enhancers or promoters. We use embryonic stem cells (ESCs), which contain a full spectrum of active and repressed (poised) enhancers, to train predictive models. As many poised enhancers in ESCs switch towards an active state during differentiation, predictive models can also be trained on poised enhancers throughout differentiation and in development. Remarkably, we determine that histone modifications at enhancers, as well as promoters, are predictive of gene expression in ESCs and throughout differentiation and development. Importantly, we demonstrate that their contribution to the predictive models varies depending on their location in enhancers or promoters. Moreover, we use a local regression (LOESS) to normalize sequencing data from different sources, which allows us to apply predictive models trained in a specific cellular context to a different one. We conclude that the relationship between gene expression and histone modifications at enhancers is universal and different from promoters. Our study provides new insight into how histone modifications relate to gene expression based on their location in enhancers or promoters.

Activating RNAs associate with Mediator to enhance chromatin architecture and transcription.

Recent advances in genomic research have revealed the existence of a large number of transcripts devoid of protein-coding potential in multiple organisms. Although the functional role for long non-coding RNAs (lncRNAs) has been best defined in epigenetic phenomena such as X-chromosome inactivation and imprinting, different classes of lncRNAs may have varied biological functions. We and others have identified a class of lncRNAs, termed ncRNA-activating (ncRNA-a), that function to activate their neighbouring genes using a cis-mediated mechanism. To define the precise mode by which such enhancer-like RNAs function, we depleted factors with known roles in transcriptional activation and assessed their role in RNA-dependent activation. Here we report that depletion of the components of the co-activator complex, Mediator, specifically and potently diminished the ncRNA-induced activation of transcription in a heterologous reporter assay using human HEK293 cells. In vivo, Mediator is recruited to ncRNA-a target genes and regulates their expression. We show that ncRNA-a interact with Mediator to regulate its chromatin localization and kinase activity towards histone H3 serine 10. The Mediator complex harbouring disease- displays diminished ability to associate with activating ncRNAs. Chromosome conformation capture confirmed the presence of DNA looping between the ncRNA-a loci and its targets. Importantly, depletion of Mediator subunits or ncRNA-a reduced the chromatin looping between the two loci. Our results identify the human Mediator complex as the transducer of activating ncRNAs and highlight the importance of Mediator and activating ncRNA association in human disease.

DPY30 regulates pathways in cellular senescence through ID protein expression.

Cellular senescence is an intrinsic defense mechanism to various cellular stresses: while still metabolically active, senescent cells stop dividing and enter a proliferation arrest. Here, we identify DPY30, a member of all mammalian histone H3K4 histone methyltransferases (HMTases), as a key regulator of the proliferation potential of human primary cells. Following depletion of DPY30, cells show a severe proliferation defect and display a senescent phenotype, including a flattened and enlarged morphology, elevated level of reactive oxygen species (ROS), increased SA-ß-galactosidase activity, and formation of senescence-associated heterochromatin foci (SAHFs). While DPY30 depletion leads to a reduced level of H3K4me3-marked active chromatin, we observed a concomitant activation of CDK inhibitors, including p16INK4a, independent of H3K4me3. ChIP experiments show that key regulators of cell-cycle progression, including ID proteins, are under direct control of DPY30. Because ID proteins are negative regulators of the transcription factors ETS1/2, depletion of DPY30 leads to the transcriptional activation of p16INK4a by ETS1/2 and thus to a senescent-like phenotype. Ectoptic re-introduction of ID protein expression can partially rescue the senescence-like phenotype induced by DPY30 depletion. Thus, our data indicate that DPY30 controls proliferation by regulating ID proteins expression, which in turn lead to senescence bypass.

How ribosomes make peptide bonds.

Ribosomes are molecular machines that synthesize proteins in the cell. Recent biochemical analyses and high-resolution crystal structures of the bacterial ribosome have shown that the active site for the formation of peptide bonds--the peptidyl-transferase center--is composed solely of rRNA. Thus, the ribosome is the largest known RNA catalyst and the only natural ribozyme that has a synthetic activity. The ribosome employs entropic catalysis to accelerate peptide-bond formation by positioning substrates, reorganizing water in the active site and providing an electrostatic network that stabilizes reaction intermediates. Proton transfer during the reaction seems to be promoted by a concerted shuttle mechanism that involves ribose hydroxyl groups on the tRNA substrate.

Peptide bond formation does not involve acid-base catalysis by ribosomal residues.

Ribosomes catalyze the formation of peptide bonds between aminoacyl esters of transfer RNAs within a catalytic center composed of ribosomal RNA only. Here we show that the reaction of P-site formylmethionine (fMet)-tRNA(fMet) with a modified A-site tRNA substrate, Phelac-tRNA(Phe), in which the nucleophilic amino group is replaced with a hydroxyl group, does not show the pH dependence observed with small substrate analogs such as puromycin and hydroxypuromycin. This indicates that acid-base catalysis by ribosomal residues is not important in the reaction with the full-size substrate. Rather, the ribosome catalyzes peptide bond formation by positioning the tRNAs, or their 3' termini, through interactions with rRNA that induce and/or stabilize a pH-insensitive conformation of the active site and provide a preorganized environment facilitating the reaction. The rate of peptide bond formation with unmodified Phe-tRNA(Phe) is estimated to be >300 s(-1).

Modulating the activity of the peptidyl transferase center of the ribosome.

The peptidyl transferase (PT) center of the ribosome catalyzes two nucleophilic reactions, peptide bond formation between aminoacylated tRNA substrates and, together with release factor, peptide release. Structure and function of the PT center are modulated by binding of aminoacyl-tRNA or release factor, thus providing the basis for the specificity of catalysis. Another way by which the function of the PT center is controlled is signaling from the peptide exit tunnel. The SecM nascent peptide induces ribosome stalling, presumably by inhibition of peptide bond formation. Similarly, the release factor-induced hydrolytic activity of the PT center can be suppressed by the TnaC nascent peptide contained in the exit tunnel. Thus, local and long-range conformational rearrangements can lead to changes in the reaction specificity and catalytic activity of the PT center.

Exploration of the conserved A+C wobble pair within the ribosomal peptidyl transferase center using affinity purified mutant ribosomes.

Protein synthesis in the ribosome's large subunit occurs within an active site comprised exclusively of RNA. Mutational studies of rRNA active site residues could provide valuable insight into the mechanism of peptide bond formation, but many of these mutations cause a dominant lethal phenotype, which prevents production of the homogeneous mutant ribosomes needed for analysis. We report a general method to affinity purify in vivo assembled 50S ribosomal subunits containing lethal active site mutations via a U1A protein-binding tag inserted onto the 23S rRNA. The expected pH-dependent formation of the A2450+C2063 wobble pair has made it a potential candidate for the pH-dependent conformational change that occurs within the ribosomal active site. Using this approach, the active site A2450+C2063 pair was mutated to the isosteric, but pH-independent, G2450*U2063 wobble pair, and 50S subunits containing the mutations were affinity purified. The G*U mutation caused the adjacent A2451 to become hyper-reactive to dimethylsulfate (DMS) modification in a pH-independent manner. Furthermore, the G*U mutation decreased both the rate of peptide bond formation and the affinity of the post-translocation complex for puromycin. The reaction rate (k(pep)) was reduced approximately 200-fold for both puromycin and the natural aminoacyl-tRNA A-site substrate. The mutations also substantially altered the pH dependence of the reaction. Mutation of this base pair has significant deleterious effects upon peptidyl transferase activity, but because G*U mutation disrupts several tertiary contacts with the wobble pair, the assignment of A2450 as the active site residue with the neutral pK(a) important for the peptidyl transferase reaction cannot be fully supported or excluded based upon these data.

Role of PRC2-associated factors in stem cells and disease.

The Polycomb group (PcG) of proteins form chromatin-binding complexes with histone-modifying activity. The two main PcG repressive complexes studied (PRC1 and PRC2) are generally associated with chromatin in its repressed state. PRC2 is responsible for methylation of histone H3 at lysine 27 (H3K27me3), an epigenetic mark that is linked with numerous biological processes, including development, adult homeostasis and cancer. The core canonical complex PRC2, which contains the EZH1/2, SUZ12 and EED proteins, may be extended and functionally manipulated through interactions with several other proteins. In this review, we focus on these PRC2-associated proteins. As PRC2 functions are diverse, the variability conferred by these sub-stoichiometrically associated members may help to understand specific changes in PRC2 activity, chromatin recruitment and distribution required for gene repression.

Modulation of the rate of peptidyl transfer on the ribosome by the nature of substrates.

The ribosome catalyzes peptide bond formation between peptidyl-tRNA in the P site and aminoacyl-tRNA in the A site. Here, we show that the nature of the C-terminal amino acid residue in the P-site peptidyl-tRNA strongly affects the rate of peptidyl transfer. Depending on the C-terminal amino acid of the peptidyl-tRNA, the rate of reaction with the small A-site substrate puromycin varied between 100 and 0.14 s(-1), regardless of the tRNA identity. The reactivity decreased in the order Lys = Arg > Ala > Ser > Phe = Val > Asp >> Pro, with Pro being by far the slowest. However, when Phe-tRNA(Phe) was used as A-site substrate, the rate of peptide bond formation with any peptidyl-tRNA was approximately 7 s(-1), which corresponds to the rate of binding of Phe-tRNA(Phe) to the A site (accommodation). Because accommodation is rate-limiting for peptide bond formation, the reaction rate is uniform for all peptidyl-tRNAs, regardless of the variations of the intrinsic chemical reactivities. On the other hand, the 50-fold increase in the reaction rate for peptidyl-tRNA ending with Pro suggests that full-length aminoacyl-tRNA in the A site greatly accelerates peptide bond formation.

The ribosomal peptidyl transferase.

Peptide bond formation on the ribosome takes place in an active site composed of RNA. Recent progress of structural, biochemical, and computational approaches has provided a fairly detailed picture of the catalytic mechanism of the reaction. The ribosome accelerates peptide bond formation by lowering the activation entropy of the reaction due to positioning the two substrates, ordering water in the active site, and providing an electrostatic network that stabilizes the reaction intermediates. Proton transfer during the reaction appears to be promoted by a concerted proton shuttle mechanism that involves ribose hydroxyl groups on the tRNA substrate.

Importance of tRNA interactions with 23S rRNA for peptide bond formation on the ribosome: studies with substrate analogs.

The major enzymatic activity of the ribosome is the catalysis of peptide bond formation. The active site -- the peptidyl transferase center -- is composed of ribosomal RNA (rRNA), and interactions between rRNA and the reactants, peptidyl-tRNA and aminoacyl-tRNA, are crucial for the reaction to proceed rapidly and efficiently. Here, we describe the influence of rRNA interactions with cytidine residues in A-site substrate analogs (C-puromycin or CC-puromycin), mimicking C74 and C75 of tRNA on the reaction. Base-pairing of C75 with G2553 of 23S rRNA accelerates peptide bond formation, presumably by stabilizing the peptidyl transferase center in its productive conformation. When C74 is also present in the substrate analog, the reaction is slowed down considerably, indicating a slow step in substrate binding to the active site, which limits the reaction rate. The tRNA-rRNA interactions lead to a robust reaction that is insensitive to pH changes or base substitutions in 23S rRNA at the active site of the ribosome.

Mechanism of peptide bond formation on the ribosome.

Peptide bond formation is the fundamental reaction of ribosomal protein synthesis. The ribosome's active site--the peptidyl transferase center--is composed of rRNA, and thus the ribosome is the largest known RNA catalyst. The ribosome accelerates peptide bond formation by 10(7)-fold relative to the uncatalyzed reaction. Recent progress of structural, biochemical and computational approaches has provided a fairly detailed picture of the catalytic mechanisms employed by the ribosome. Energetically, catalysis is entirely entropic, indicating an important role of solvent reorganization, substrate positioning, and/or orientation of the reacting groups within the active site. The ribosome provides a pre-organized network of electrostatic interactions that stabilize the transition state and facilitate proton shuttling involving ribose hydroxyl groups of tRNA. The catalytic mechanism employed by the ribosome suggests how ancient RNA-world enzymes may have functioned.

Rapid peptide bond formation on isolated 50S ribosomal subunits.

The catalytic site of the ribosome, the peptidyl transferase centre, is located on the large (50S in bacteria) ribosomal subunit. On the basis of results obtained with small substrate analogues, isolated 50S subunits seem to be less active in peptide bond formation than 70S ribosomes by several orders of magnitude, suggesting that the reaction mechanisms on 50S subunits and 70S ribosomes may be different. Here we show that with full-size fMet-tRNA(fMet) and puromycin or C-puromycin as peptide donor and acceptor substrates, respectively, the reaction proceeds as rapidly on 50S subunits as on 70S ribosomes, indicating that the intrinsic activity of 50S subunits is not different from that of 70S ribosomes. The faster reaction on 50S subunits with fMet-tRNA(fMet), compared with oligonucleotide substrate analogues, suggests that full-size transfer RNA in the P site is important for maintaining the active conformation of the peptidyl transferase centre.

Essential mechanisms in the catalysis of peptide bond formation on the ribosome.

Peptide bond formation is the main catalytic function of the ribosome. The mechanism of catalysis is presumed to be highly conserved in all organisms. We tested the conservation by comparing mechanistic features of the peptidyl transfer reaction on ribosomes from Escherichia coli and the Gram-positive bacterium Mycobacterium smegmatis. In both cases, the major contribution to catalysis was the lowering of the activation entropy. The rate of peptide bond formation was pH independent with the natural substrate, amino-acyl-tRNA, but was slowed down 200-fold with decreasing pH when puromycin was used as a substrate analog. Mutation of the conserved base A2451 of 23 S rRNA to U did not abolish the pH dependence of the reaction with puromycin in M. smegmatis, suggesting that A2451 did not confer the pH dependence. However, the A2451U mutation alters the structure of the peptidyl transferase center and changes the pattern of pH-dependent rearrangements, as probed by chemical modification of 23 S rRNA. A2451 seems to function as a pivot point in ordering the structure of the peptidyl transferase center rather than taking part in chemical catalysis.

The ribosome as an entropy trap.

To determine the effectiveness of the ribosome as a catalyst, we compared the rate of uncatalyzed peptide bond formation, by the reaction of the ethylene glycol ester of N-formylglycine with Tris(hydroxymethyl)aminomethane, with the rate of peptidyl transfer by the ribosome. Activation parameters were also determined for both reactions, from the temperature dependence of their second-order rate constants. In contrast with most protein enzymes, the enthalpy of activation is slightly less favorable on the ribosome than in solution. The 2 x 10(7)-fold rate enhancement produced by the ribosome is achieved entirely by lowering the entropy of activation. These results are consistent with the view that the ribosome enhances the rate of peptide bond formation mainly by positioning the substrates and/or water exclusion within the active site, rather than by conventional chemical catalysis.

The G2447A mutation does not affect ionization of a ribosomal group taking part in peptide bond formation.

Peptide bond formation on the ribosome is catalyzed by RNA. Kinetic studies using Escherichia coli ribosomes have shown that catalysis (>10(5)-fold overall acceleration) is due to a large part to substrate positioning. However, peptide bond formation is inhibited approximately 100-fold by protonation of a ribosomal group with pKa=7.5, indicating either a contribution of general acid-base catalysis or inhibition by a pH-dependent conformational change within the active site. The function of a general base has been attributed to A2451 of 23S rRNA, and a charge relay system involving G2447 has been postulated to bring about the extensive pKa shift of A2451 implied in the model. Using a rapid kinetic assay, we found that the G2447A mutation, which has essentially no effect on cell growth, lowers the rate of peptide bond formation about 10-fold and does not affect the ionization of the ribosomal group with pKa=7.5 taking part in the reaction. This result does not support the proposed charge relay mechanism involving G2447 and the role of A2451 as general base in the catalysis of peptide bond formation.