Title and Abstract, Sussex July 10^th 2012

Oscar Llorca

Centre for Biological Research (CIB), Spanish National Research Council (CSIC), Madrid

Transient geometries in nonsense-mediated mRNA decay (NMD) by cryo-EM

The structural analysis of short-lived macromolecular interactions by cryo-EM is challenged by the tendency of these complexes to fall apart under the conditions used in EM. These complexes can also display flexible/transient conformations, which, if undetected and/or adequately handled, can result in solving “incorrect” structures. I will describe our experience undertaking the structural analysis of transient interactions and conformations, including glutaraldehyde cross-linking, increasing affinity by introducing mutations and classification of conformational transitions co-existing in the same sample by image processing. I will describe examples on several on-going projects in the group.

A big part of my talk will deal with our recent work on nonsense-mediated mRNA decay (NMD) (Arias-Palomo et al. Genes Dev. 2011; Melero et al. Nat Struct Mol Biol. 2012). NMD is a post-transcriptional surveillance mechanism that recognizes and degrades mRNAs containing premature translation termination codons (PTCs). In humans, NMD is essential and elicited when a stop codon is present upstream of an exon-junction-complex (EJC). The crosstalk between a ribosome and an EJC is mediated by a collection of NMD factors that interact forming transient complexes. Despite intensive research, the molecular mechanisms that determine that an aberrant mRNA is tagged for degradation are not completely understood, partly due to the difficulty obtaining structural information of intermediates in large macromolecular complexes.

We have produced a complex corresponding to an intermediate step during NMD and containing seven different proteins. The complex was stable at high concentrations but it disassembled at the concentrations used in cryo-EM. Thus, the complex was stabilized using GraFix and its cryo-electron microscopy structure solved at 16 Å resolution. To position the different proteins and propose a pseudo-atomic model of the complex, we carried out negative-stain reconstructions of the complex lacking specific subunits, experiments to locate the 3' and 5' ends of the RNA in the complex and mass spectrometry to identify regions involved in protein-protein interactions.

Short CV

Oscar Llorca obtained his Ph.D. in Molecular Biology in 1996 at the National Centre for Biotechnology (CNB) in Madrid (Spain) working on the structure of GroEL using electron microscopy under the supervision of JM Valpuesta and JL Carrascosa. During a first post-doc in Madrid he characterised actin and tubulin folding mediated by the eukaryotic cytosolic chaperonin by cryo-EM. In 2000 he moved to the Chester Beatty Laboratories (Institute of Cancer Research, London, UK) as EU Marie Curie Fellow in Keith R. Willison´s group and in collaboration with Alan Ashworth to study the structure of DNA repair complexes. In June 2002 he moved to the Centre for Biological Research (CIB) of the Spanish National Research Council in Madrid, setting up his own group dedicated to the structural and functional analysis of large macromolecular complexes using electron microscopy. The group devotes to the functional and structural understanding of large complexes in DNA and RNA transactions (DNA repair, DNA replication and mRNA decay) and complexes that regulate the complement in the innate immune system.

Selected recent publications:

A. Macromolecular complexes in DNA and RNA transactions

Melero R, Buchwald G, Castaño R, Raabe M, Gil D, Lázaro M, Urlaub H, Conti E, Llorca O. The cryo-EM structure of the UPF-EJC complex shows UPF1 poised toward the RNA 3´ end. Nat Struct Mol Biol. 2012 19, 498–505 (2012).

Arias-Palomo E et al., Ohno S and Llorca O. Nonsense-Mediated mRNA Decay SMG-1 kinase is regulated by large-scale conformational changes controlled by SMG-8. Genes & Development. 2011 Jan 15;25(2):153-64.

Núñez-Ramírez R, Klinge S, Sauguet L, Melero R, Recuero-Checa MA, Kilkenny M, Perera RL, García-Alvarez B, Hall RJ, Nogales E, Pellegrini L, Llorca O. Flexible tethering of primase and DNA Pol {alpha} in the eukaryotic primosome. Nucleic Acids Res. 2012

Fernández IS et al., Ohno S, Llorca O. Characterization of SMG-9, an essential component of the nonsense-mediated mRNA decay SMG1C complex. Nucleic Acids Res. 2011 Jan 1;39(1):347-58.

Klinge S, Núñez-Ramírez R, Llorca O, Pellegrini L. 3D architecture of DNA Pol alpha reveals the functional core of multi-subunit replicative polymerases. EMBO Journal. 2009 Jul 8;28(13):1978-87.

Torreira E et al., Ayora S, Dutta A, Llorca O. Architecture of the pontin/reptin complex, essential in the assembly of several macromolecular complexes. Structure. 2008 Oct 8;16(10):1511-20

Adami, A., García-Alvarez, B., Arias-Palomo, E., Barford D., Llorca O. Structure of TOR and its complex with KOG1. Molecular Cell 2007 Aug 3;27(3):509-16.

Spagnolo L, Rivera-Calzada A, Pearl LH, Llorca O. Three-dimensional structure of the human DNA-PKcs/Ku70/Ku80 complex assembled on DNA and its implications for DNA DSB repair. Molecular Cell 2006 May 19;22(4):511-9.

Rivera-Calzada A, Maman JD, Spagnolo L, Pearl LH, Llorca O. Three-dimensional structure and regulation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Structure. 2005 Feb;13(2):243-55.

B. Structural immunology of complement regulation

Hadders MA, Bubeck D, Roversi P, Hakobyan S, Forneris F, Morgan BP, Pangburn MK, Llorca O, Lea SM, Gros P. Assembly and Regulation of the Membrane Attack Complex Based on Structures of C5b6 and sC5b9. Cell Rep. 2012 Mar 29;1:1-8. 

Alcorlo M, Martínez-Barricarte R, Fernández FJ, Rodríguez-Gallego C, Round A, Vega MC, Harris CL, de Cordoba SR, Llorca O. Unique structure of iC3b resolved at a resolution of 24 A by 3D-electron microscopy. Proc Natl Acad Sci U S A. 2011 Aug 9;108(32):13236-40.

de Córdoba SR, Harris CL, Morgan BP, Llorca O. Lessons from functional and structural analyses of disease-associated genetic variants in the complement alternative pathway. Biochim Biophys Acta-Molecular Basis of Disease. 2011 Jan;1812(1):12-22.

Torreira E, Tortajada A, Montes T, Rodríguez de Córdoba S, Llorca O. 3D structure of the C3bB complex provides insights into the activation and regulation of the complement alternative pathway convertase. Proc Natl Acad Sci U S A. 2009 Jan 20;106(3):882-7.

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Last updated: Wednesday, 16 May 2012