Internal Ribosome Entry Site (IRES) advances and applications

Welwyn Garden City, Hertfordshire
Tuesday, 23 October 2007

Internal Ribosome Entry Site (IRES) advances and applications

Internal Ribosome Entry Site (IRES) advances and applications
Tuesday, 23 October 2007 09:00 - 17:00

BioPark Hertfordshire
Broadwater Road
Welwyn Garden City
Hertfordshire
AL7 3AX
United Kingdom

Map and Directions

Protein synthesis can be considered as a three-stage process, consisting of initiation, elongation and termination. It is the final stage in the process of gene expression and involves numerous cellular proteins and RNAs. The initiation step is the primary focus for regulation. Translation initiation on the majority of cellular mRNAs is mediated by a mechanism involving recognition of the 5’ terminal cap-structure (m7GpppN..). However, a different mechanism, termed internal initiation, was first shown to occur on certain uncapped viral mRNAs (e.g. from picornaviruses). An internal ribosome entry site (IRES) element present within the 5’ untranslated region (UTR) of the viral RNA directs initiation of protein synthesis at an internal position hundreds of nucleotides downstream from the 5’ terminus. These IRES elements are highly structured and position the ribosome at, or just upstream of, the initiation codon. IRES elements have now also been found in certain cellular mRNAs; these generally encode proteins involved in apoptosis, cell cycle regulation or stress responses. In the past few years much has been discovered about how these IRES elements function-including their structures, RNA-RNA interactions and the involvement of cellular proteins in their action. This meeting will bring together experts studying the mechanism of action of IRES elements and their role in translational control of viral and cellular protein synthesis, to review the latest research in this area. In addition, there will also be a focus on the biotechnological exploitation of these elements, for example in gene therapy and protein expression vectors.

Chair: Dr Lisa Roberts , Senior Lecturer in Virology
School of Biomedical and Molecular Sciences
University of Surrey

9:00 – 9:45 Registration

9:45 – 10:00 Introduction by the Chair: Dr Lisa Roberts, University of Surrey, UK

10:00 – 10:30 Picornavirus IRES elements; variations on a theme

Dr Graham Belsham, National Veterinary Institute, DTU, Denmark

IRES elements were first characterized within the RNAs from various picornaviruses including poliovirus, human rhinoviruses, encephalomyocarditis virus and foot-and-mouth disease virus. These represent examples of two different types of IRES element. However, it is now known that there are 4 different classes of picornavirus IRES element, these differ in size, secondary (and presumably tertiary) structure and their requirement for cellular translation initiation factors plus other trans-acting factors. Remarkably, one of these types of picornavirus IRES element is very similar to the IRES elements found within hepatitis C virus and other members of the flaviviridae (e.g. the pestiviruses).

10:30 – 11:00 The role of RNA structure in picornavirus IRES activity

Encarna Martinez Salas, Centro de Biologia Molecular, Madrid, Spain

Understanding of internal ribosome entry site (IRES) function requires a detailed knowledge of each step involved in the internal initiation process, from RNA folding and IRES-protein interaction to ribosome recruitment. Thus, deciphering the IRES accessibility to external agents due to RNA structural features as well as RNA-protein protections within living cells is of primary importance. The accessibility of unpaired nucleotides in the entire foot-and-mouth disease (FMDV) IRES has been obtained in vitro by RNA probing techniques; subsequently it has been used to interpret the footprint data obtained in vivo for the mRNA encompassing the IRES element in the intercistronic space. Two chemical reagents, dimethyl-sulfate and AMT-psoralen, that enters the cell membrane and interact with nucleic acids in a structure-dependent manner have been used to footprint the FMDV IRES in living cells in the context of a biologically active mRNA. The results of DMS accessibility and UV-psoralen crosslinking in the competitive cellular environment evidenced differences in RNA structure with data obtained in vitro, and provided essential

11:00- 11:10 Group and speakers photo

11:10 – 11:30 Mid-morning break

11:30 – 12:00 Structural and functional analysis of IRES trans-acting factors
Dr Stephen Curry, Imperial College, UK

The IRES elements of picornaviruses display a requirement for a diverse array of host-cell RNA binding proteins, known as IRES trans-acting factors (ITAFs), many of which are not normally involved in translation initiation. The mechanism of action of these ITAFs is poorly understood, largely because structural studies of IRES-protein complexes present stiff challenges to investigators. Our group has focused on elucidating the solution and crystal structures of a number of distinct ITAFs, including PTB, La and ITAF45. These studies have provided many valuable insights and are laying important groundwork for future investigations. Recent highlights will be summarised.

12:00 – 12:30 Trans-acting factors of cellular and artificial IRESes

Dr Keith Spriggs, Nottingham University, UK

12:30 – 12:50 Tour of the BioPark

12:50 – 14:00 Lunch and Poster Viewing

14:00 – 14:30 Effect of oxidative stress on HCV IRES-mediated and host cell translation.

Dr Shiu-WanChan, University of Manchester, UK

Hepatitis C patients very often present elevated levels of oxidants in the blood and liver. Thus, the potential of anti-oxidants in anti-HCV therapy has been recognized and compounds of this type are now in clinical trials. Before we can fully appreciate the potential of anti-oxidants in anti-HCV therapy we must understand the effects of oxidative stress on the virus. HCV utilises an IRES element for translation, in contrast to cap-dependent translation of the majority of cellular proteins. To understand how virus translation is modulated under oxidative stress, we compared HCV IRES-mediated translation with cap-dependent translation using a bicistronic reporter construct.

14:30 – 15:00 IRES-mediated translational regulation of FGF-1 expression during myogenesis and muscle

regeneration

Dr Caroline Conte, Institut National de la santé et de la recherche médicale, France

FGF-1 is expressed as a single protein, under the control of four distinct promoters allowing synthesis of four transcripts. We identified IRESs in transcripts A, B and C. We studied the regulation of FGF-1 expression in differentiating myoblasts and in a mouse model of muscular regeneration. Promoter A and the IRES A are activated concomitantly with expression of endogenous FGF-1. These results reveal a unique co-regulation of promoter and IRES, suggesting a mechanism of coupled translation and transcription. In addition, IRES A is an excellent candidate to be used in multicistronic vectors aimed to co-express therapeutic molecules against myopathies

15:00 – 15:30 Afternoon Tea/Coffee and Last Poster Viewing

15:30 – 16:00 Structural and biochemical studies of the HCV IRES

Dr. Peter Lukavsky, MRC LMB, UK

IRES RNAs from the hepatitis C virus and classical swine fever virus coordinate cap-independent assembly of eukaryotic 48S initiation complexes. Here we report that these IRESes also play a functional role during 80S ribosome assembly downstream of 48S complex formation, in promoting eIF5-induced GTP hydrolysis and eIF2/GDP release from the initiation complex. We show that this function is encoded in their independently folded IRES domain II and that it depends both on its characteristic bent conformation and conserved RNA motifs. Our data suggest a general mode of subunit joining in HCV and HCV-like IRESes

16:00 – 16:30 Insect virus IRES elements-functional analysis and utility in protein expression systems

Dr Lisa Roberts, University of Surrey, UK

We have previously demonstrated that the 5' untranslated region of the Rhopalosiphum padi virus (RhPV) genome contains a novel internal ribosome entry site (IRES) element that functions in mammalian, insect and plant in vitro translation systems. We have recently defined the minimal sequences required for directing internal initiation in mammalian (RRL), plant (WGE) and insect (Sf21 cells) translation systems and have shown that the 3' unstructured region within the 5'UTR seems to be critical for IRES function. Use of this IRES element in the generation of an improved baculovirus expression vector will also be described.

16:30 – 17:00 Chairman’s summing up & close.

 

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