Factor Independent Initiation of Protein Synthesis by IRES RNAs Prof. Jeffrey S. Kieft

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1 Factor Independent Initiation Jeffrey S. Kieft, Ph.D. University of Colorado School of Medicine 1 Why is initiation so important? Initiation Elongation Fidelity translation needs to start at the right spot on the mrna Termination Rapidity how quickly the needed proteins are made Regulation initiation is the primary phase for regulation of translation Ribosome recycling 2 Two ways to initiate in eukaryotes Cap-dependent, scanning-dependent mechanism Cap-independent, internal initiation of translation driven by internal ribosome entry sites (IRESes) 3 The screen versions of these slides have full details of copyright and acknowledgements 1

2 Outline of the lecture What is an IRES RNA? What are the specific RNA structures that drive IRES function? What do we know about the structure-based mechanism of IRES function? 4 Eukaryotic translation initiation In the mrna: 5 and 3 UTR structure, protein binding Polyaden ylation uorfs Recoding signals IRESes Initiation factors: eif4e Recruitment eif2 of the ribosome to the 4E-BPs mrna begins with eif4g recognition of the 7-methyl guanosine cap by eif4e Ribosome: Other: S6 phosphorylation mirnas Other modification s 5 A simple view of ribosome recruitment in eukaryotes 6 The screen versions of these slides have full details of copyright and acknowledgements 2

3 Internal ribosome entry sites (IRESes) Cap dependent Cap independent IRES RNA 7 Positive strand ssrna viruses Picornaviridae Human poliovirus 1 Human coxsackievirus B3 Bovine enterovirus Human rhinovirus Hepatitis A virus EMCV FMDV Equine rhinitis virus Human echovirus Porcine teschovirus 1 CRPV-like (insect picorna-like) PSIV HiPV CrPV RHpV BQV, etc. Flaviviridae Bovine viral diarrhea virus Classical swine fever virus Hepatitis C virus GB virus B Tobamovirus Crucifer tobamovirus Potyviridae Tobacco etch virus Some viral IRESes RNA reverse transcribing viruses Retroviridae SIV HIV 1 Hyman T-lymphotropic virus type 1 Moloney murine leukemia virus Friend murine leukemia virus Harvey murine sarcoma virus Avian reticuloendotheliosis virus Murine leukemia virus Rous sarcoma virus dsdna viruses Herpesviridae Kaposi s sarcoma-associated herpesvirus 8 Example of a single-stranded RNA virus replication cycle (HCV) 5 RNA (+/-) 3 5 RNA (+) 3 Golgi RNP Complex Nucleus 9 The screen versions of these slides have full details of copyright and acknowledgements 3

4 Some cellular IRESes Transcription factors Antennapedia Ultrabithorax C-myc N-myc Myt2 AMLI/RUNX1 Gtx C-Jun Mnt Nkx6.1 NRF YAP1 Smad5 HIF-1 alpha Hairless RNA processing factors La autoantigen Translation Factors TIF4631 eif4gi P97/DAP5/NAT1 Stress response XIAP APC Apaf-1 BAG-1 Bip/GRP78 Cellular communication /interactions FGF2 PDGF-2/c-sis VEGF-A IGF-II Estrogen receptor alpha IGF-1 receptor Notch2 Connexin 43, 32 Cyr61 Cytoskeleton associated proteins ARC MAP2 Kinases Pim-1 P58 PITSLRE Alpha-Cam kinase II CDK inhibitor p27 Protein kinase Cdelta Channels/Transporters KV.14 Beta F1-ATPase Cat-1 Misc. ODC Dendrin Neurogranin/RC3 NBS1 FMR1 Rbm3 NDST 10 Outline of the lecture What is an IRES RNA? What are the specific RNA structures that drive IRES function? What do we know about the structure-based mechanism of IRES function? 11 Viral IRES mechanistic classes Class I direct ribosome binding Class II uses a subset of the canonical initiation factors Class III uses several canonical factors and one or more IRES Trans-Activating Factors (ITAFs) 12 The screen versions of these slides have full details of copyright and acknowledgements 4

5 Three IRES RNA secondary structures Class II Class III Class I 13 Cryo-EM reconstructions of IRES-40S complexes Class II Class I HCV IRES- 40S From: Spahn & Kieft et al. Hepatitis C virus IRES RNA-induced changes in the conformation of the 40s ribosomal subunit, Science 291, (2001) CrPV IRES-40S From: Spahn, C. M. et al.. Cryo-EM visualization of a viral internal ribosome entry site bound to human ribosomes; The IRES functions as an RNA-based translation factor, Cell 118, (2004) 14 Ribosome manipulation by the HCV IRES From: Spahn & Kieft et al., Hepatitis C virus IRES RNA-induced changes in the conformation of the 40s ribosomal subunit Science 291, (2001) 15 The screen versions of these slides have full details of copyright and acknowledgements 5

6 Viral IRES mechanistic classes Class I direct ribosome binding Class II uses a subset of the canonical initiation factors Class III uses several canonical factors and one or more IRES Trans-Activating Factors (ITAFs) 16 Secondary structure of a Dicistroviridae IGR IRES Upstream ORF Start site 17 The Dicistroviridae IGR IRES: a manipulator of the translation apparatus J. E. Wilson, T. V. Pestova, C. U. Hellen, P. Sarnow, (2000) J. Sasaki, N. Nakashima, (1999) and (2000) 18 The screen versions of these slides have full details of copyright and acknowledgements 6

7 Hypothesis The IGR IRES RNA s function depends on a specific three-dimensional fold that allows it to interact specifically with and manipulate the ribosome Questions Does the IGR IRES have a higher-order folded structure? If so, what is the structural basis for ribosome binding and manipulation by this all-rna initiation apparatus? 19 Hydroxyl radical probing of PSIV IRES Costantino & Kieft (2005) RNA 11: Conservation among the IGR IRES folds Conservation among the IGR IRES folds 21 The screen versions of these slides have full details of copyright and acknowledgements 7

8 Domains 1 and 2 come The IGR IRES architecture together to form a compact structure (the ribosome binding domain) Domain 3 is not part of the compact structure formed by the other two domains, but is placed into the P-site of the mrna decoding groove of the ribosome Ribosome binding domain Kanamori & Nakashima (2001) Jan & Sarnow (2002) Y.T. Nishiyama et al. (2003) Costantino & Kieft (2005) P-site domain 22 Architecture of the IRES P-Site Domain Ribosome Binding Domain Kanamori & Nakashima (2001) Jan & Sarnow (2002) T. Nishiyama et al. (2003) Costantino & Kieft (2005) Spahn et al. (2004) 23 Solving the structure of two domains Ribosome bindingdomain Resolution = 3.1Å P63 a = b = 132Å, c = 160Å Rwork = 25.3% Rfree = 29.4% Phased using MAD signal from iridium (III) hexammine P-site domain 24 The screen versions of these slides have full details of copyright and acknowledgements 8

9 The crystal structure of the ribosome binding domain of the PSIV IGR IRES From: Pfingsten, J.S., Costantino, D.A., & Kieft, J.S. (2006) Science 341: The crystal structure is consistent with solution probing data Costantino & Kieft (2005) Also: mutagenesis, native gels, analytical ultracentrifug ati on, enzymatic probing, directed hydroxyl radical probing 26 Specific contacts between the ribosome and the IRES RNA rps25 From: Pfingsten, J.S., Costantino, D.A., & Kieft, J.S. (2006) Science 341: The screen versions of these slides have full details of copyright and acknowledgements 9

10 Analysis of mutants confirms interaction with 60S subunit From: Pfingsten, J.S., Costantino, D.A., & Kieft, J.S. (2006) Science 341: Conserved bases in the IRES structure Conserved bases in the IRES structure Alignment from: Kanamori & Nakashima, (2001) 29 Differing mechanisms extend to after 80S formation J. E. Wilson, T. V. Pestova, C. U. Hellen, P. Sarnow, (2000) J. Sasaki, N. Nakashima, (1999) and (2000) 30 The screen versions of these slides have full details of copyright and acknowledgements 10

11 Structures of both domains were solved Ribosome binding domain P-site domain Resolution = 3.1Å P63 a = b = 132Å, c = 160Å Rwork = 25.3% Rfree = 29.4% Phased using MAD signal from iridium (III) hexammine Resolution = 2.4Å P41212 a= b = 59.5Å, c = 99.8Å Rwork = 24.5% Rfree = 27.2% Solved using MAD with cobalt and iridium hexammine 31 Structure of domain 3 / P-site domain of CrPV Costantino, D.A., Pfingsten, J.S. Rambo, R.P. & Kieft, J.S. submitted 32 The pseudoknot mimics a codon-anticodon interaction trna- mrna: Selmer, et al. (2006) Structure of the 70S Ribosome Complexed with mrna and trna Science 313: The screen versions of these slides have full details of copyright and acknowledgements 11

12 Interactions that stabilize the structure Iridium (III) hexammine 34 Costantino, D.A., Pfingsten, J.S. Rambo, R.P. & Kieft, J.S. submitted Translation assays of IRES mutants Costantino, D.A., Pfingsten, J.S. Rambo, R.P. & Kieft, J.S. submitted 35 The complete IRES on the 40S subunit Cryo-EM from Spahn et al. (2004) Cell 36 The screen versions of these slides have full details of copyright and acknowledgements 12

13 Outline of the lecture What is an IRES RNA? What are the specific RNA structures that drive IRES function? What do we know about the structure-based mechanism of IRES function? 37 Docking the structure into the structure of the ribosome+trna+mrna Red = CrPV domain 3 Gray = P-site initiator trna Selmer et al. (2006) Structure of the 70S Ribosome Complexed with mrna and trna Science 313: Positional similarity between this IRES domain and a P/E hybrid state trna trna structures/positions at right from: Allen, G. S. et al. (2005) The cryo-em structure of a translation initiation complex from Escherichia coli, Cell 121, The screen versions of these slides have full details of copyright and acknowledgements 13

14 The complete IRES on the ribosome 40 Hypothesized trna hybrid state mimicry Canonical translocation during elongation Proposed hybrid state mimicry eef2+ GTP 41 A complete and testable mechanistic model 42 The screen versions of these slides have full details of copyright and acknowledgements 14

15 Conclusion By studying this example of a factor independent RNA-based translation apparatus, w e gain insight into the mechanism of this class I IRES So, w hat can this tell us about other IRESes (and RNA-base translation regulation in general)? 43 Cryo-EM reconstructions of IRES-40S complexes Class II Class I HCV IRES- 40S From: Spahn & Kieft et al. Hepatitis C virus IRES RNA-induced changes in the conformation of the 40s ribosomal subunit, Science 291, (2001) CrPV IRES-40S From: Spahn, C. M. et al. Cryo-EM Visualization of a Viral Internal Ribosome Entry Site Bound to Human Ribosomes; The IRES Functions as an RNA-Based Translation Factor Cell 118, (2004) 44 Comparison of this IRES with that from hepatitis C virus rps5 IRES structural changes 210 L1 stalk 40S-bound Spahn and Kieft et al. (2001) Science 291 pp Model of 48S-bound Siridechadilok, B., et al. (2005) Science, 310, pp S-bound Boehringer et al. (2005) Structure 13, pp The screen versions of these slides have full details of copyright and acknowledgements 15

16 NMR and crystal structures of HCV IRES domains 46 Reviewing the lecture What is an IRES RNA? IRES RNAs are specific RNA sequences that drive translation initiation in the absence of a cap IRES RNAs are critical for the infection of many viruses and may be critical players in gene regulation in cells IRES RNAs have diverse sequences and requirement s for protein factors 47 Reviewing the lecture cont. What are the specific RNA structures that drive IRES function? The folds of IRES RNAs are diverse some are loose collections of secondary structure elements, and some are compact folds In the case of the Dicistrovirid ae IGR IRESes, the structure appears to mimic parts of trna Parts of the HCV IRES structure have been solved, but more structural information about this IRES, and other IRESes is needed 48 The screen versions of these slides have full details of copyright and acknowledgements 16

17 Reviewing the lecture cont. What do w e know about the structure-based mechanism of IRES function? Combined use of cryo-em, biochemical, functional, and structural data is leading to a model for Dicistroviridae IGR IRES function trna mimicry may be a key part of IRES function To what degree different IRESes have mechanistic similarities remains unclear more experimentation is needed The screen versions of these slides have full details of copyright and acknowledgements 17