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Structural and functional studies of argonaute and tomato aspermy virus protein 2B, a suppressor of RNAi

  • RNA interference (RNAi) is triggered by recognition of double-stranded RNA (dsRNA), and elicits the silencing of gene(s) complementary to the dsRNA sequence. RNAi is thought to have emerged as a way of safeguarding the genome against mobile genetic elements and viral infection, thus maintaining genomic integrity. dsRNA is first processed into small interfering RNAs (siRNA) by the enzyme Dicer. siRNAs are ~21 to 25 -nt long, and contain a signature 5’ phosphate group and a two nucleotide long 3’ overhang (Bernstein et al., 2001). The siRNA is then loaded into the RNA-induced si-lencing complex (RISC), of which Argonaute is the primary catalytic component (Liu et al., 2004). Energetic asymmetry of the siRNA ends allows for its directional loading into RISC (Khvorova et al., 2003; Schwarz et al., 2003). Argonaute cleaves the passen-ger strand of the siRNA, leaving the guide strand of the siRNA bound to RISC (Gregory et al., 2005; Matranga et al., 2005; Rand et al., 2005). This single-stranded guide strand siRNA bound to Argonaute is able to recognize target mRNA in a sequence-specific manner, and cleaves the mRNA. Argonaute 2 in complex with single-stranded siRNA is sufficient for mRNA recognition and cleavage, thus forming a minimal RISC (Rivas et al., 2005). miRNAs, endogenously expressed small RNA genes which typically contain mismatches and non-Watson-Crick base pairing, are processed by this general pathway, although typically modulate gene expression by translational repression as opposed to cleavage of their target mRNA. The number of Argonaute genes is highly variable between species, ranging from one in S. pombe to twenty-seven in C. elegans. Earlier crystal structures of Argonaute apoen-zymes show the architecture of Argonaute to be a multidomain protein composed of N terminal, PAZ, MID, and PIWI domains (Song et al., 2004; Yuan et al., 2005). These multi-domain proteins are present in both prokaryotic and eukaryotic organisms. The role of Argonaute proteins in prokaryotes is still unknown, but based similarity to eu-karyotic Argonautes, they may also be involved in nucleic acid-directed regulatory pathways. These proteins have served as excellent models for learning about the struc-ture and function of this family of proteins. RNAi has found a widespread application for the simple yet effective knockdown of genes of interest. The catalytic cycle of RISC requires the binding of a number of different nucleotide structures to Argonaute, and we expect Argonaute to undergo a number of conforma-tional changes during the cycle of mRNA recognition by RISC (Filipowicz, 2005; Tom-ari and Zamore, 2005). Nevertheless, it remains unclear how the multi-domain ar-rangement of Argonaute recognizes and distinguishes between single-stranded and dou-ble-stranded oligonucleotides, which correspond to the Dicer-processed siRNA product, guide strand siRNA, and the guide strand / mRNA duplex. The Argonaute protein from Aquifex aeolicus was cloned, expressed, crystallized and solved by molecular replacement. Relative to earlier Argonaute structures, a 24° reorientation of the PAZ domain in this structure opens a basic cleft between the N-terminal and PAZ domains, exposing the guide strand binding pocket of PAZ. A 5.5-ns molecular dynamics simulation of Argonaute showed a strong tendency of the PAZ and N-terminal domains to be mobile. Binding of single-stranded DNA to Argonaute was monitored by total internal reflection fluorescence spectroscopy (TIRFS). The experi-ments showed biphasic kinetics indicative of large conformational changes, and re-vealed a hotspot of binding energy corresponding to the first 9 nucleotides, the so-called “seed region” most crucial for sequence-specific target recognition. As RNAi may have evolved as a way of safeguarding the genome viral infection, it is not surprising that viruses have evolved different strategies to suppress the host RNAi response in the form of viral suppressor protein. (Hock and Meister, 2008; Lecellier and Voinnet, 2004; Rashid et al., 2007; Song et al., 2004; Vastenhouw and Plasterk, 2004). These viral suppressors are widespread, having been identified in a number of different viral families. Not surprisingly, they generally share little sequence homology with one another, although they appear to exist as oligomers built upon a ~ 100-200 amino acid protomer. Tomato aspermy virus, a member of the Cucumoviruses, encodes for protein 2B (TAV 2B, 95 a.a., ~11.3 kDa) that acts as an RNAi suppressor. Intriguingly, a similar genomic arrangement is seen in RNAi suppressors in the Nodaviruses, a family of viruses that can infect both plants and animals, such as Flock house virus b2 (FHV b2). The 2B and b2 proteins are both derived from a frameshifted ORF within the RNA polymerase gene (Chao et al., 2005). In spite of this genomic similarity, the 2B and b2 proteins share little sequence identity, and it is not well understood how the Cucumovirus 2B proteins suppress RNAi. To address how TAV 2B suppresses RNAi, the oligonucleotide-binding properties of TAV 2B were studied. TAV 2B shows a preference for double-stranded RNA oligonucleotides corresponding to siRNAs and miRNAs, and also binds to single-stranded RNA oligonucleotides. A stretch of positively charged residues between amino acids 20-30 are critical for RNA binding. Binding to RNA oligomerizes and induces a conformational change in TAV 2B into a primarily helical structure. These studies sug-gest that suppression of RNAi by TAV 2B may occur by targeting different stages of the RNAi pathway. TAV 2B falls under the category of more general RNAi suppres-sors, with potentially multiple targets for suppression.
  • RNA Interferenz (RNAi) ist ein hochkonservierter Mechanismus, der dem silencing von Genen dient. Dieser Prozess findet in den meisten Eukaryoten statt und wird durch die Gegenwart exogener, in die Zelle aufgenommener oder endogener, doppelsträngiger RNA (dsRNA) eingeleitet. Das Vorhandensein dieser dsRNA resultiert in einer sequenzspezifischen Degradation der Ziel-mRNA (Sontheimer, 2005; Tomari & Zamore, 2005; Voinnet, 2005). RNA silencing wurde zufällig in Organismen entdeckt, die transgene DNA trugen, mit exogener DNA behandelt wurden oder mit Virus infiziert waren. Etliche Gen silencing Phänomene auf posttranskriptioneller Ebene wurden in Pflanzen, Pilzen, Tieren und Wimperntierchen entdeckt und stellten somit die Basis für das Konzept des posttranskriptionellen Gen silencing (PTGS) oder RNA silencing dar. RNA silencing wurde zuerst in Pflanzen beobachtet, als Wissenschaftler versuchten, eine stärker violettfarbene Petunie zu generieren. Dazu kreierten sie eine transgene Pflanze, die eine Extra-Kopie des Gens für das Enzym trug, das für die violette Pigmentation verantwortlich ist. Überraschenderweise wies der Abkömmling keine intensivere violette Färbung auf, im Gegenteil, seine Blüten waren weiß. Sowohl die Produktion der transgenen, als auch der Wildtyp Pigmentationsgene der violetten Petunie wurden ausgeschaltet oder co-suppressiert (Napoli et al., 1990; Smith et al., 2000; van der Krol et al., 1990). PTGS kann demnach auch Transgene beeinflussen, die nicht homolog zu endogenen Genen sind, was darauf schließen lässt, dass dieser Prozess keineswegs ein simpler Regulationsmechanismus für die Kontrolle endogener Gene darstellt (Dehio & Schell, 1994; Ingelbrecht et al., 1994). Ein ähnliches Phänomen wurde im Pilz Neurospora crassa als „quelling“ bezeichnet. In diesem Pilz resultierte die Einführung homologer RNA-Sequenzen in einer Degradation endogener Gene (Romano & Macino, 1992). In C. elegans wurden die ersten Erkenntnisse über ein RNA vermitteltes silencing durch die Einführung von Antisense- oder Sense-RNA erhalten. Diese RNA war komplementär zur mRNA des Ziel-Gens. Die Experimente wurden mit der Idee durchgeführt, dass die Antisense-RNA einen Doppelstrang mit seiner komplementären mRNA bilden soll und folglich die Translation zum Protein blockiert wurde (Guo & Kemphues, 1995). 1998 zeigten Wissenschaftler in C. elegans, dass ein silencing der Expression exo- und endogener Gene durch die Injektion von komplementärer dsRNA stattfand. Dieses Phänomen wurde als RNA Interferenz bezeichnet (Fire et al., 1998). ....

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Metadaten
Author:Umar Jan Rashid
URN:urn:nbn:de:hebis:30-71403
Referee:Julian Chen, Beatrix SüßGND
Advisor:Julian Chen
Document Type:Doctoral Thesis
Language:English
Year of Completion:2008
Year of first Publication:2008
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Granting Institution:Johann Wolfgang Goethe-Universität
Release Date:2009/10/02
Page Number:137
Note:
Diese Dissertation steht außerhalb der Universitätsbibliothek leider (aus urheberrechtlichen Gründen) nicht im Volltext zur Verfügung, die CD-ROM kann (auch über Fernleihe) bei der UB Frankfurt am Main ausgeliehen werden.
HeBIS-PPN:417688326
Institutes:Biochemie, Chemie und Pharmazie / Biochemie und Chemie
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Sammlungen:Sammlung Biologie / Weitere biologische Literatur (eingeschränkter Zugriff)
Licence (German):License LogoArchivex. zur Lesesaalplatznutzung § 52b UrhG