Chen Davidovich

Promiscuous RNA binding by Polycomb repressive complex 2

Polycomb repressive complex 2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. Long noncoding RNAs (lncRNAs) recruit PRC2 to chromatin, but the general role of RNA in maintaining repressed chromatin is unknown. Here we measure the binding constants of human PRC2 to various RNAs and find comparable affinity for human lncRNAs targeted by PRC2 as for irrelevant transcripts from ciliates and bacteria. PRC2 binding is size dependent, with lower affinity for shorter RNAs. In vivo, PRC2 predominantly occupies repressed genes; PRC2 is also associated with active genes, but most of those are not regulated by PRC2. These findings support a model in which PRC2s promiscuous binding to RNA transcripts allows it to scan for target genes that have escaped repression, thus leading to maintenance of the repressed state. Such RNAs may also provide a decoy for PRC2.

Induced-fit tightens pleuromutilins binding to ribosomes and remote interactions enable their selectivity

New insights into functional flexibility at the peptidyl transferase center (PTC) and its vicinity were obtained by analysis of pleuromutilins binding modes to the ribosome. The crystal structures of Deinococcus radiodurans large ribosomal subunit complexed with each of three pleuromutilin derivatives: retapamulin (SB-275833), SB-280080, and SB-571519, show that all bind to the PTC with their core oriented similarly at the A-site and their C14 extensions pointing toward the P-site. Except for an H-bond network with a single nucleotide, G2061, which involves the essential keto group of all three compounds, only minor hydrophobic contacts are formed between the pleuromutilin C14 extensions and any ribosomal component, consistent with the PTC tolerance to amino acid diversity. Efficient drug binding mode is attained by a mechanism based on induced-fit motions exploiting the ribosomal intrinsic functional flexibility and resulting in conformational rearrangements that seal the pleuromutilin-binding pocket and tightens it up. Comparative studies identified a network of remote interactions around the PTC, indicating that pleuromutilins selectivity is acquired by nonconserved nucleotides residing in the PTC vicinity, in a fashion resembling allosterism. Likewise, pleuromutilin resistant mechanisms involve nucleotides residing in the environs of the binding pocket, consistent with their slow resistance-development rates.

RNA Duplex Map in Living Cells Reveals Higher-Order Transcriptome Structure

RNA has the intrinsic property to base pair, forming complex structures fundamental to its diverse functions. Here, we develop PARIS, a method based on reversible psoralen crosslinking for global mapping of RNA duplexes with near base-pair resolution in living cells. PARIS analysis in three human and mouse cell types reveals frequent long-range structures, higher-order architectures, and RNA-RNA interactions in trans across the transcriptome. PARIS determines base-pairing interactions on an individual-molecule level, revealing pervasive alternative conformations. We used PARIS-determined helices to guide phylogenetic analysis of RNA structures and discovered conserved long-range and alternative structures. XIST, a long noncoding RNA (lncRNA) essential for X chromosome inactivation, folds into evolutionarily conserved RNA structural domains that span many kilobases. XIST A-repeat forms complex inter-repeat duplexes that nucleate higher-order assembly of the key epigenetic silencing protein SPEN. PARIS is a generally applicable and versatile method that provides novel insights into the RNA structurome and interactome. VIDEO ABSTRACT.

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.

The recruitment of chromatin modifiers by long noncoding RNAs: lessons from PRC2

Polycomb repressive complex-2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. Among chromatin modifying factors shown to be recruited and regulated by long noncoding RNAs (lncRNAs), PRC2 is one of the most studied. Mammalian PRC2 binds thousands of RNAs in vivo, and it is becoming a model system for the recruitment of chromatin modifying factors by RNA. Yet, well-defined PRC2-binding motifs within target RNAs have been elusive. From the protein side, PRC2 RNA-binding subunits contain no known RNA-binding domains, complicating functional studies. Here we provide a critical review of existing models for the recruitment of PRC2 to chromatin by RNAs. This discussion may also serve researchers who are studying the recruitment of other chromatin modifiers by lncRNAs.

The evolving ribosome: from non-coded peptide bond formation to sophisticated translation machinery

Structural analysis supported by biochemical, mutagenesis and computational evidence, revealed that the contemporary ribosomes active site is a universal symmetrical pocket made of ribosomal RNA. This pocket seems to be the remnant of the proto-ribosome, a dimeric RNA assembly evolved by gene duplication, capable of autonomously catalyzing peptide bond formation and non-coded amino acid polymerization.

Structural basis for cross-resistance to ribosomal PTC antibiotics

Clinically relevant antibiotics that target the ribosomal peptidyl transferase center (PTC), a highly conserved ribosomal region, exert their inhibitory action by exploiting the flexibility of PTC nucleotides, which trigger modulations of the shape of the antibiotic binding pocket. Resistance to these antibiotics was observed clinically and in vitro. Based on the crystal structures of the large ribosomal subunit from eubacterium suitable to represent pathogens in complex with these antibiotics, it was found that all nucleotides mediating resistance to PTC antibiotics cluster on one side of the PTC. Over half of the nucleotides affecting resistance reside in regions of lower sequence conservation, and are too distal to make Van der Waals interactions with the bound drugs. Alterations of the identity of these nucleotides may not lethally affect ribosome function, but can hamper antibiotic binding through changes in the conformation and flexibility of specific PTC nucleotides. Comparative analysis revealed properties likely to lead to cross-resistance and enabled their parameterization. As the same nucleotides are frequently involved in resistance to more than a single family of antibiotics, the common pattern explains medically observed cross-resistance to PTC antibiotics and suggests the potential for a wider clinical threat.

Ancient machinery embedded in the contemporary ribosome

Structural analysis, supported by biochemical, mutagenesis and computational evidence, indicates that the peptidyltransferase centre of the contemporary ribosome is a universal symmetrical pocket composed solely of rRNA. This pocket seems to be a relic of the proto-ribosome, an ancient ribozyme, which was a dimeric RNA assembly formed from self-folded RNA chains of identical, similar or different sequences. This could have occurred spontaneously by gene duplication or gene fusion. This pocket-like entity was capable of autonomously catalysing various reactions, including peptide bond formation and non-coded or semi-coded amino acid polymerization. Efforts toward the structural definition of the early entity capable of genetic decoding involve the crystallization of the small ribosomal subunit of a bacterial organism harbouring a single functional rRNA operon.

The structure of ribosome-lankacidin complex reveals ribosomal sites for synergistic antibiotics

Crystallographic analysis revealed that the 17-member polyketide antibiotic lankacidin produced by Streptomyces rochei binds at the peptidyl transferase center of the eubacterial large ribosomal subunit. Biochemical and functional studies verified this finding and showed interference with peptide bond formation. Chemical probing indicated that the macrolide lankamycin, a second antibiotic produced by the same species, binds at a neighboring site, at the ribosome exit tunnel. These two antibiotics can bind to the ribosome simultaneously and display synergy in inhibiting bacterial growth. The binding site of lankacidin and lankamycin partially overlap with the binding site of another pair of synergistic antibiotics, the streptogramins. Thus, at least two pairs of structurally dissimilar compounds have been selected in the course of evolution to act synergistically by targeting neighboring sites in the ribosome. These results underscore the importance of the corresponding ribosomal sites for development of clinically relevant synergistic antibiotics and demonstrate the utility of structural analysis for providing new directions for drug discovery.

Ribosome's mode of function: myths, facts and recent results†

Ribosomes translate the genetic code into proteins in all living cells with extremely high efficiency, owing to their inherent flexibility and to their spectacular architecture. During the last 6 decades, extensive effort has been made to elucidate the molecular mechanisms associated with their function, and a quantum jump has been made in recent years, once the three dimensional structures of ribosomes and their functional complexes have been determined. These illuminated key issues in ribosome function, confirmed various biochemical, genetic, and medical findings, and revealed mechanistic details beyond previous expectation, thus leading to conceptual revolutions, and turning old myths into actual facts. Copyright