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Internal Ribosome Entry Sites

    Internal ribosome entry sites (IRESs) are RNA sequences that can recruit the translation machinery independent of the 5' end of the messenger RNA. IRESs are found in both viral and cellular RNAs and are important for regulating gene
    expression. There is great diversity in the mechanisms used by IRESs to recruit the ribosome and this is reflected in a variety of RNA sequences that function as IRESs. The ability of an RNA sequence to function as an IRES is conferred by
    structures operating at multiple levels from primary sequence through higher order three-dimensional structures within dynamic ribonucleoproteins (RNPs).

    IRES are likely to be present in conditions where cap-dependent translation is inhibited

    • mitosis
    • viral infection, especially picornavirus
    • hypoxia

    or in viruses where m7G cap is absent (picornavirus,and?)


    • monocistronic
    • bicistronic
    • false positives/negatives
      • cryptic promoter
      • cryptic splicing
      • unstable transcrpt/transfected RNA

    Characteristics of IRES

    • No difference in GC content from non IRES UTR (Baird et al., 2006)
    • UTR longer than average ~150 base (Baird et al., 2006)
    • viral regions with IRES tend to be longer 190 - 450 bases
    • generally, but not always, upstream of initiation codon

    Follow up on


    • 1.  Balvay L, Soto Rifo R, Ricci EP, Decimo D, Ohlmann T. Structural and functional diversity of viral IRESes. Biochim Biophys Acta 2009, 1789:542–557.
    • 2.  Gilbert WV, Zhou K, Butler TK, Doudna JA. Cap independent translation is required for starvation induced differentiation in yeast. Science 2007, 317:1224–1227.
      IRES includes 60 base "unstructured" tract with PAB as ITAF
    • 3.  E. Martınez-Salas, A. Pacheco, P. Serrano, and N. Fernandez, “New insights into internal ribosome entry site elements relevant for viral gene expression,” Journal of General Virology, vol. 89, no. 3, pp. 611–626, 2008.
    • 4.  G. Hern´andez, “Was the initiation of translation in early eukaryotes IRES-driven?” Trends in Biochemical Sciences, vol. 33, no. 2, pp. 58–64, 2008.

    Secondary structure - how much is really known ?


    • 5.  Shibuya N, Nakashima N. Characterization of the 5� internal ribosome entry site of Plautia stali intestine virus. J Gen Virol 2006, 87:3679–3686.
    • 6.  Sasaki J, Nakashima N. Translation initiation at the CUU codon is mediated by the internal ribosome entry site of an insect picorna-like virus in vitro. J Virol 1999, 73:1219–1226.
    • 7.  Kanamori Y, Nakashima N. A tertiary structure model of the internal ribosome entry site (IRES) for methionine-independent initiation of translation.RNA 2001, 7:266–274.
    • 8.  Hellen CU, de Breyne S. A distinct group of hepacivirus/pestivirus-like internal ribosomal entry sites in members of diverse picornavirus genera: evidence for modular exchange of functional noncoding RNA elements by recombination. J Virol 2007, 81:5850–5863.
    • 9.  Lukavsky PJ. Structure and function of HCV IRES domains. Virus Res 2009, 139:166–171.
    • 10. Fraser CS, Hershey JW, Doudna JA. The pathway of hepatitis C virus mRNA recruitment to the human ribosome. Nat Struct Mol Biol 2009, 16:397–404.
    • 11.  Easton LE, Locker N, Lukavsky PJ. Conserved functional domains and a novel tertiary interaction near the pseudoknot drive translational activity of hepatitis C virus and hepatitis C virus-like internal ribosome entry sites. Nucleic Acids Res 2009, 37:5537–5549.
    • 12.  Morris MJ, Negishi Y, Pazsint C, Schonhoft JD, Basu S. An RNA G-quadruplex is essential for capindependent translation initiation in human VEGF IRES. J Am Chem Soc 2010, 132:17831–17839.
    Plank, "deletion analysis of cellular IRESs indicates that small fragments of RNA are capable of initiating internal translation initiation as well, or sometimes better, than the full-length 5' UTR."
    • 13.  Meng Z, Jackson NL, Shcherbakov OD, Choi H, Blume SW. The human IGF1R IRES likely operates through a Shine-Dalgarno-like interaction with the G961 loop (E-site) of the 18S rRNA and is kinetically modulated by a naturally polymorphic polyU loop. J Cell Biochem 2010, 110:531–544.
    • 14.  Chappell SA, Edelman GM, Mauro VP. A 9-nt segment of a cellular mRNA can function as an internal ribosome entry site (IRES) and when present in linked multiple copies greatly enhances IRES activity. Proc Natl Acad Sci U S A 2000, 97:1536–1541.
    • 15.  Gilbert WV, Zhou K, Butler TK, Doudna JA. Capindependent translation is required for starvationinduced differentiation in yeast. Science 2007, 317:1224–1227.
    Plank "Evidence suggests that distal regions of RNA within the IRES interact directly, indicative of a higher-order fold that is important for IRES function"
    • 16.  Serrano P, Ramajo J, Martinez-Salas E. Rescue of internal initiation of translation by RNA complementation provides evidence for a distribution of functions between individual IRES domains. Virology 2009, 388:221–229.
    • 17.  Fernandez-Miragall O, Ramos R, Ramajo J, Martinez- Salas E. Evidence of reciprocal tertiary interactions between conserved motifs involved in organizing RNA structure essential for internal initiation of translation. RNA 2006, 12:223–234.
    • 18.  Ramos R, Martinez-Salas E. Long-range RNA interactions between structural domains of the aphthovirus internal ribosome entry site (IRES). RNA 1999, 5:1374–1383.
    • 19.  Yu Y, Abaeva IS, Marintchev A, Pestova TV, Hellen CU. Common conformational changes induced in type 2 picornavirus IRESs by cognate trans-acting factors. Nucleic Acids Res 2011, 39:4851–4865.

    PSIV-IGR Pseudoknots (refs from Plank, not sure they are on the topic, per se)

    • 20.  Pfingsten JS, Castile AE, Kieft JS. Mechanistic role of structurally dynamic regions in Dicistroviridae IGR IRESs. J Mol Biol 2010, 395:205–217.
    • 21.  Jang CJ, Lo MC, Jan E. Conserved element of the dicistrovirus IGR IRES that mimics an E-site tRNA/ribosome interaction mediates multiple functions. J Mol Biol 2009, 387:42–58.
    • 22.  Costantino DA, Pfingsten JS, Rambo RP, Kieft JS. tRNA-mRNA mimicry drives translation initiation from a viral IRES. Nat Struct Mol Biol 2008, 15:57–64.
    • 23.  Costantino D, Kieft JS. A preformed compact ribosome-binding domain in the cricket paralysis-like
      virus IRES RNAs. RNA 2005, 11:332–343.
    • 24.  Nishiyama T, Yamamoto H, Shibuya N, Hatakeyama Y, Hachimori A, Uchiumi T, Nakashima N. Structural elements in the internal ribosome entry site of Plautia stali intestine virus responsible for binding with ribosomes. Nucleic Acids Res 2003, 31:2434–2442.

    3D structures

    • 25. Filbin ME, Kieft JS. Toward a structural understanding of IRES RNA function. Curr Opin Struct Biol 2009, 19:267–276.
    • 26. Kieft JS. Viral IRES RNA structures and ribosome interactions. Trends Biochem Sci 2008, 33:274–283.
    • 27. Spahn CM, Jan E,Mulder A, Grassucci RA, Sarnow P, Frank J. Cryo-EM visualization of a viral internal ribosome entry site bound to human ribosomes; the IRES functions as an RNA-based translation factor. Cell 2004, 118:465–475.
    • 28. Schuler M, Connell SR, Lescoute A, Giesebrecht J, Dabrowski M, Schroeer B, Mielke T, Penczek PA, Westhof E, Spahn CM. Structure of the ribosomebound cricket paralysis virus IRES RNA. Nat Struct Mol Biol 2006, 13:1092–1096.
    • 29. Boehringer D, Thermann R, Ostareck-Lederer A, Lewis JD, Stark H. Structure of the hepatitis C Virus IRES bound to the human 80S ribosome: remodeling of the HCV IRES. Structure (Camb) 2005, 13:1695–1706.
    • 30. Zhu J, Korostelev A, Costantino DA, Donohue JP, Noller HF, Kieft JS. Crystal structures of complexes containing domains from two viral internal ribosome entry site (IRES) RNAs bound to the 70S ribosome. Proc Natl Acad Sci U S A 2011, 108:1839–1844.
    • 31. Pfingsten JS, Costantino DA, Kieft JS. Structural basis for ribosome recruitment and manipulation by a viral IRES RNA. Science 2006, 314:1450–1454.
    • 32. Lukavsky PJ, Kim I, Otto GA, Puglisi JD. Structure of HCV IRES domain II determined by NMR. Nat Struct Biol 2003, 10:1033–1038.
    • 33. Collier AJ, Gallego J, Klinck R, Cole PT, Harris SJ, Harrison GP, Aboul-Ela F, Varani G, Walker S. A conserved RNA structure within the HCV IRES eIF3- binding site. Nat Struct Biol 2002, 9:375–380.
    • 34. Lukavsky PJ, Otto GA, Lancaster AM, Sarnow P, Puglisi JD. Structures of two RNA domains essential for hepatitis C virus internal ribosome entry site function. Nat Struct Biol 2000, 7:1105–1110.
    • 35. Kieft JS, Zhou K, Grech A, Jubin R, Doudna JA. Crystal structure of an RNA tertiary domain essential to HCV IRES-mediated translation initiation. Nat Struct Biol 2002, 9:370–374.
    • 36. Zhao Q, Han Q, Kissinger CR, Hermann T, Thompson PA. Structure of hepatitis C virus IRES subdomain IIa. Acta Crystallogr D Biol Crystallogr 2008, 64:436–443.
    • 37. Z. Wang, M. Parisien, K. Scheets, and W. A. Miller, “The cap-binding translation initiation factor, eIF4E, binds a pseudoknot in a viral cap-independent translation element,” Structure, vol. 19, pp. 868–880, 2011.

    3' elements

    • 38. A. M. Rakotondrafara, C. Polacek, E. Harris, and W. A. Miller, “Oscillating kissing stem-loop interactions mediate 5� scanning-dependent translation by a viral 3�-capindependent translation element,” RNA, vol. 12, no. 10, pp. 1893–1906, 2006.
    • 39. V. A. Stupina, X. Yuan, A. Meskauskas, J. D. Dinman, and A. E. Simon, “Ribosome binding to a 5� translational enhancer is altered in the presence of the 3� untranslated region in capindependent translation of turnip crinkle virus,” Journal of Virology, vol. 85, pp. 4638–4653, 2011.
    • 40. X. Yuan, K. Shi, A. Meskauskas, and A. E. Simon, “The 3� end of Turnip crinkle virus contains a highly interactive structure including a translational enhancer that is disrupted by binding to the RNA-dependent RNA polymerase,” RNA, vol. 15, no. 10, pp. 1849–1864, 2009.


    • 41. Komar AA, Hatzoglou M. Cellular IRES-mediated translation: the war of ITAFs in pathophysiological states. Cell Cycle 2011, 10:229–240.
    • 42. Lewis SM, Holcik M. For IRES trans-acting factors, it is all about location. Oncogene 2008, 27:1033–1035.
    • 43. Stoneley M, Willis AE. Cellular internal ribosome entry segments: structures, trans-acting factors and regulation of gene expression. Oncogene 2004, 23:3200–3207.
    • 44. King HA, Cobbold LC, Willis AE. The role of IRES trans-acting factors in regulating translation initiation. Biochem Soc Trans 2010, 38:1581–1586.
    • 45. Spriggs KA, Bushell M, Mitchell SA, Willis AE. Internal ribosome entry segment-mediated translation during apoptosis: the role of IRES-trans-acting factors. Cell Death Differ 2005, 12:585–591.
    • 46. Mitchell SA, Brown EC, Coldwell MJ, Jackson RJ, Willis AE. Protein factor requirements of the Apaf-1 internal ribosome entry segment: roles of polypyrimidine tract binding protein and upstream of N-ras. Mol Cell Biol 2001, 21:3364–3374.
    • 47. Pickering BM, Mitchell SA, Spriggs KA, Stoneley M, Willis AE. Bag-1 internal ribosome entry segment activity is promoted by structural changes mediated by poly(rC) binding protein 1 and recruitment of polypyrimidine tract binding protein 1. Mol Cell Biol 2004, 24:5595–5605.
    • 48. Kafasla P, Morgner N, Poyry TA, Curry S, Robinson CV, Jackson RJ. Polypyrimidine tract binding protein stabilizes the encephalomyocarditis virus IRES structure via binding multiple sites in a unique orientation. Mol Cell 2009, 34:556–568.
    • 49. A. Pacheco and E. Martinez-Salas, “Insights into the biology of IRES elements through riboproteomic approaches,” Journal of Biomedicine and Biotechnology, vol. 2010, Article ID 458927, 12 pages, 2010.
    • 50. A. Pacheco, S. Reigadas, and E. Mart´ınez-Salas, “Riboproteomic analysis of polypeptides interacting with the internal ribosome-entry site element of foot-and-mouth disease viral RNA,” Proteomics, vol. 8, no. 22, pp. 4782–4790, 2008.

    Computational methods

    • 51. Hatakeyama Y, Shibuya N, Nishiyama T, Nakashima N. Structural variant of the intergenic internal ribosome entry site elements in dicistroviruses and computational search for their counterparts. RNA 2004, 10:779–786.
    • 52. Baird SD, Turcotte M, Korneluk RG, Holcik M. Searching for IRES. RNA 2006, 12:1755–1785.
    • 53. Baird SD, Lewis SM, Turcotte M, Holcik M. A search for structurally similar cellular internal ribosome entry sites. Nucleic Acids Res 2007, 35:4664–4677.
    • 54. Mokrejs M, Masek T, Vopalensky V, Hlubucek P, Delbos P, PospisekM. IRESite–a tool for the examination of viral and cellular internal ribosome entry sites. Nucleic Acids Res 2010, 38:D131–D136. IRESite
    • 55. Low JT, Weeks KM. SHAPE-directed RNA secondary structure prediction. Methods 2010, 52:150–158.
    • 56. T. Y.Wu, C. C. Hsieh, J. J. Hong, C. Y. Chen, and Y. S. Tsai, “IRSS: a web-based tool for automatic layout and analysis of IRES secondary structure prediction and searching system in silico,” BMC Bioinformatics, vol. 10, article 160, 2009.
    • 57. Klein, R. J., & Eddy, S. R. (2003). RSEARCH: finding homologs of single structured RNA sequences. Bmc Bioinformatics, 4(1), 44.
    • 58. Fernández, N., Fernandez-Miragall, O., Ramajo, J., García-Sacristán, A., Bellora, N., Eyras, E., ... & Martínez-Salas, E. (2011). Structural basis for the biological relevance of the invariant apical stem in IRES-mediated translation. Nucleic acids research, 39(19), 8572-8585.
    • 59. Fernández-Miragall, O., & Martínez-Salas, E. (2003). Structural organization of a viral IRES depends on the integrity of the GNRA motif. Rna, 9(11), 1333-1344.
    • 60. Bergamini, G. I. O. V. A. N. N. A., Preiss, T. H. O. M. A. S., & Hentze, M. W. (2000). Picornavirus IRESes and the poly (A) tail jointly promote cap-independent translation in a mammalian cell-free system. Rna, 6(12), 1781-1790.
    • 61. Plank, T. D. M., & Kieft, J. S. (2012). The structures of nonprotein‐coding RNAs that drive internal ribosome entry site function. Wiley Interdisciplinary Reviews: RNA, 3(2), 195-212.
    • 62. Paek, K. Y., Park, S. M., Hong, K. Y., & Jang, S. K. (2012). Cap-dependent translation without base-by-base scanning of an messenger ribonucleic acid. Nucleic Acids Research, 40(15), 7541-7551.
    • 63. Malys, N., & McCarthy, J. E. (2011). Translation initiation: variations in the mechanism can be anticipated. Cellular and Molecular Life Sciences, 68(6), 991-1003.
    • 64. Martínez-Salas, E., Piñeiro, D., & Fernández, N. (2012). Alternative mechanisms to initiate translation in eukaryotic mRNAs. Comparative and Functional Genomics, 2012.
    • 65. Komar, A. A., Mazumder, B., & Merrick, W. C. (2012). A new framework for understanding IRES-mediated translation. Gene.
    • 66. Landry, D. M., Hertz, M. I., & Thompson, S. R. (2009). RPS25 is essential for translation initiation by the Dicistroviridae and hepatitis C viral IRESs. Genes & development, 23(23), 2753-2764.
    • 67. Allam, H., & Ali, N. (2010). Initiation factor eIF2-independent mode of c-Src mRNA translation occurs via an internal ribosome entry site. Journal of Biological Chemistry, 285(8), 5713-5725.
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