The Tudor domain of human PHF1 recognizes trimethylated lysine 36 of histone H3 (H3K36me3). This interaction modulates methyltransferase activity of the PRC2 complex and plays a role in retention of PHF1 at the DNA damage sites. We have previously determined the structural basis for the association of Tudor with a methylated histone peptide. Here we detail the molecular mechanism of binding of the Tudor domain to the H3KC36me3-nucleosome core particle (H3KC36me3-NCP). Using a combination of TROSY NMR and FRET we show that Tudor concomitantly interacts with H3K36me3 and DNA. Binding of the PHF1 Tudor domain to the H3KC36me3-NCP stabilizes the nucleosome in a conformation in which the nucleosomal DNA is more accessible to DNA-binding regulatory proteins. Our data provide a mechanistic explanation for the consequence of reading of the active mark H3K36me3 by the PHF1 Tudor domain.

The initial transcript of the GLS1 gene undergoes alternative splicing to produce two glutaminase variants (KGA and GAC) that contain unique C-terminal sequences. A truncated form of human glutaminase (hGA(124-551)) that lacks either C-terminal sequence was expressed in E.Coli and purified. This construct exhibits a hyperbolic glutamine saturation profile (K(m) of 1.6 mM). BPTES, bis-2[5-phenylacetamido-1,2,4-thiadiazol-2-yl]ethylsulfide, functions as a potent uncompetitive inhibitor of this construct (K(i) of 0.2 µM). The hGA(124-551) is inactive in the absence of phosphate, but exhibits a hyperbolic phosphate-dependent activation profile that is also inhibited by BPTES. Gel filtration studies indicate that hGA(124-551) forms a dimer in the absence or presence of 100 mM phosphate, whereas addition of BPTES causes the formation of an inactive tetramer. The combined data indicate that BPTES inhibits human glutaminase by a novel mechanism and that BPTES is a potential lead compound for development of an effective cancer chemotherapeutic agent.

Human Polycomb Repressive Complex 2 (PRC2) catalysis of histone H3 lysine 27 methylation at certain loci depends on long noncoding RNAs (lncRNAs). Yet, in apparent contradiction, RNA is a potent catalytic inhibitor of PRC2. Here, we show that intermolecular RNA-RNA interactions between the lncRNA HOTAIR and its targets can relieve RNA inhibition of PRC2. RNA bridging is promoted by heterogeneous nuclear ribonucleoprotein B1, which uses multiple protein domains to bind HOTAIR regions via multivalent protein-RNA interactions. Chemical probing demonstrates that establishing RNA-RNA interactions changes HOTAIR structure. Genome-wide HOTAIR/PRC2 activity occurs at genes whose transcripts can make favorable RNA-RNA interactions with HOTAIR. We demonstrate that RNA-RNA matches of HOTAIR with target gene RNAs can relieve the inhibitory effect of a single lncRNA for PRC2 activity after B1 dissociation. Our work highlights an intrinsic switch that allows PRC2 activity in specific RNA contexts, which could explain how many lncRNAs work with PRC2.

Structured RNA elements, programmed RNA conformational changes, and interactions between different RNA domains underlie many modes of regulating gene expression, mandating studies to understand the foundational principles that govern these phenomena. Exploring the structured 3′ untranslated region (UTR) of a viral RNA, we discovered that different contexts of the 3′-UTR confer different abilities to enhance translation of an associated open reading frame. In one context, ribosome-induced conformational changes in a ‘sensor’ RNA domain affect a separate RNA ‘functional’ domain, altering translation efficiency. The structure of the entire 3′-UTR reveals that structurally distinct domains use a spine of continuously stacked bases and a strut-like linker to create a conduit for communication within the higher-order architecture. Thus, this 3′-UTR RNA illustrates how RNA can use programmed conformational changes to sense the translation status of an upstream open reading frame, then create a tuned functional response by communicating that information to other RNA elements.

Puromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide chains, anti-puromycin immunofluorescence enables visualization of nascent protein synthesis. A common assumption in studies of local messenger RNA translation is that the anti-puromycin staining of puromycylated nascent polypeptides in fixed cells accurately reports on their original site of translation, particularly when ribosomes are stalled with elongation inhibitors prior to puromycin treatment. However, when we attempted to implement a proximity ligation assay to detect ribosome-puromycin complexes, we found no evidence to support this assumption. We further demonstrated, using biochemical assays and live cell imaging of nascent polypeptides in mammalian cells, that puromycylated nascent polypeptides rapidly dissociate from ribosomes even in the presence of elongation inhibitors. Our results suggest that attempts to define precise subcellular translation sites using anti-puromycin immunostaining may be confounded by release of puromycylated nascent polypeptide chains prior to fixation.