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Cold Spring Harbor, NY, United States

Boguth C.A.,University of Michigan | Singh P.,University of Michigan | Singh P.,Wm Keck Structural Biology Laboratory | Huang C.-C.,University of Michigan | Tesmer J.J.G.,University of Michigan
EMBO Journal | Year: 2010

G protein-coupled receptor (GPCR) kinases (GRKs) selectively recognize and are allosterically regulated by activated GPCRs, but the molecular basis for this interaction is not understood. Herein, we report crystal structures of GRK6 in which regions known to be critical for receptor phosphorylation have coalesced to stabilize the kinase domain in a closed state and to form a likely receptor docking site. The crux of this docking site is an extended N-terminal helix that bridges the large and small lobes of the kinase domain and lies adjacent to a basic surface of the protein proposed to bind anionic phospholipids. Mutation of exposed, hydrophobic residues in the N-terminal helix selectively inhibits receptor, but not peptide phosphorylation, suggesting that these residues interact directly with GPCRs. Our structural and biochemical results thus provide an explanation for how receptor recognition, phospholipid binding, and kinase activation are intimately coupled in GRKs. © 2010 European Molecular Biology Organization | All Rights Reserved. Source

Wilusz J.E.,Massachusetts Institute of Technology | JnBaptiste C.K.,Massachusetts Institute of Technology | Lu L.Y.,Massachusetts Institute of Technology | Kuhn C.-D.,Wm Keck Structural Biology Laboratory | And 3 more authors.
Genes and Development | Year: 2012

The MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) locus is misregulated in many human cancers and produces an abundant long nuclear-retained noncoding RNA. Despite being transcribed by RNA polymerase II, the 3′ end of MALAT1 is produced not by canonical cleavage/polyadenylation but instead by recognition and cleavage of a tRNA-like structure by RNase P. Mature MALAT1 thus lacks a poly(A) tail yet is expressed at a level higher than many protein-coding genes in vivo. Here we show that the 3′ ends of MALAT1 and the MEN β long noncoding RNAs are protected from 3′-5′ exonucleases by highly conserved triple helical structures. Surprisingly, when these structures are placed downstream from an ORF, the transcript is efficiently translated in vivo despite the lack of a poly(A) tail. The triple helix therefore also functions as a translational enhancer, and mutations in this region separate this translation activity from simple effects on RNA stability or transport. We further found that a transcript ending in a triple helix is efficiently repressed by microRNAs in vivo, arguing against a major role for the poly(A) tail in microRNA-mediated silencing. These results provide new insights into how transcripts that lack poly(A) tails are stabilized and regulated and suggest that RNA triple-helical structures likely have key regulatory functions in vivo. © 2012 by Cold Spring Harbor Laboratory Press. Source

Paul S.,Cold Spring Harbor Laboratory | Kuo A.,Stanford University | Schalch T.,Cold Spring Harbor Laboratory | Schalch T.,Wm Keck Structural Biology Laboratory | And 10 more authors.
Cell Reports | Year: 2013

Chromodomain Helicase DNA binding protein 5 (. CHD5) is a tumor suppressor mapping to 1p36, a genomic region that is frequently deleted in human cancer. Although CHD5 belongs to the CHD family of chromatin-remodeling proteins, whether its tumor-suppressive role involves an interaction with chromatin is unknown. Here we report that Chd5 binds the unmodified N terminus of H3 through its tandem plant homeodomains (PHDs). Genome-wide chromatin immunoprecipitation studies reveal preferential binding of Chd5 to loci lacking the active mark H3K4me3 and also identify Chd5 targets implicated in cancer. Chd5 mutations that abrogate H3 binding are unable to inhibit proliferation or transcriptionally modulate target genes, which leads to tumorigenesis in vivo. Unlike wild-type Chd5, Chd5-PHD mutants are unable to induce differentiation or efficiently suppress the growth of human neuroblastoma in vivo. Our work defines Chd5 as an N-terminally unmodified H3-binding protein and provides functional evidence that this interaction orchestrates chromatin-mediated transcriptional programs critical for tumor suppression. © 2013 The Authors. Source

Ipsaro J.J.,Wm Keck Structural Biology Laboratory | Ipsaro J.J.,Howard Hughes Medical Institute | Joshua-Tor L.,Wm Keck Structural Biology Laboratory | Joshua-Tor L.,Howard Hughes Medical Institute
Nature Structural and Molecular Biology | Year: 2015

Since its relatively recent discovery, RNA interference (RNAi) has emerged as a potent, specific and ubiquitous means of gene regulation. Through a number of pathways that are conserved in eukaryotes from yeast to humans, small noncoding RNAs direct molecular machinery to silence gene expression. In this Review, we focus on mechanisms and structures that govern RNA silencing in higher organisms. In addition to highlighting recent advances, we discuss parallels and differences among RNAi pathways. Together, the studies reviewed herein reveal the versatility and programmability of RNA-induced silencing complexes and emphasize the importance of both upstream biogenesis and downstream silencing factors. © 2015 Nature America, Inc. All rights reserved. Source

Faehnle C.,Wm Keck Structural Biology Laboratory | Elkayam E.,Wm Keck Structural Biology Laboratory | Elkayam E.,Howard Hughes Medical Institute | Haase A.D.,Howard Hughes Medical Institute | And 2 more authors.
Cell Reports | Year: 2013

Argonautes are the central protein component in small RNA silencing pathways. Of the four human Argonautes (hAgo1-hAgo4) only hAgo2 is an active slicer. We determined the structure of hAgo1 bound to endogenous copurified RNAs to 1.75Å resolution and hAgo1 loaded with let-7 microRNA to 2.1Å. Both structures are strikingly similar to the structures of hAgo2. A conserved catalytic tetrad within the PIWI domain of hAgo2 is required for its slicing activity. Completion of the tetrad, combined with a mutation on a loop adjacent to the active site of hAgo1, results in slicer activity that is substantially enhanced by swapping in the N domain of hAgo2. hAgo3, with an intact tetrad, becomes an active slicer by swapping the N domain of hAgo2 without additional mutations. Intriguingly, the elements that make Argonaute anactive slicer involve a sophisticated interplay between the active site and more distant regions of the enzyme. © 2013 The Authors. Source

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