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Proc Natl Acad Sci U S A. Oct 1, 1996; 93(20): 11115–11120.
PMCID: PMC38293

Poly(rC) binding protein 2 binds to stem-loop IV of the poliovirus RNA 5' noncoding region: identification by automated liquid chromatography-tandem mass spectrometry.

Abstract

The 5' noncoding region of poliovirus RNA contains an internal ribosome entry site (IRES) for cap-independent initiation of translation. Utilization of the IRES requires the participation of one or more cellular proteins that mediate events in the translation initiation reaction, but whose biochemical roles have not been defined. In this report, we identify a cellular RNA binding protein isolated from the ribosomal salt wash of uninfected HeLa cells that specifically binds to stem-loop IV, a domain located in the central part of the poliovirus IRES. The protein was isolated by specific RNA affinity chromatography, and 55% of its sequence was determined by automated liquid chromatography-tandem mass spectrometry. The sequence obtained matched that of poly(rC) binding protein 2 (PCBP2), previously identified as an RNA binding protein from human cells. PCBP2, as well as a related protein, PCBP1, was over-expressed in Escherichia coli after cloning the cDNAs into an expression plasmid to produce a histidine-tagged fusion protein. Specific interaction between recombinant PCBP2 and poliovirus stem-loop IV was demonstrated by RNA mobility shift analysis. The closely related PCBP1 showed no stable interaction with the RNA. Stem-loop IV RNA containing a three nucleotide insertion that abrogates translation activity and virus viability was unable to bind PCBP2.

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  • Jackson RJ, Hunt SL, Gibbs CL, Kaminski A. Internal initiation of translation of picornavirus RNAs. Mol Biol Rep. 1994 May;19(3):147–159. [PubMed]
  • Stefano JE. Purified lupus antigen La recognizes an oligouridylate stretch common to the 3' termini of RNA polymerase III transcripts. Cell. 1984 Jan;36(1):145–154. [PubMed]
  • Meerovitch K, Pelletier J, Sonenberg N. A cellular protein that binds to the 5'-noncoding region of poliovirus RNA: implications for internal translation initiation. Genes Dev. 1989 Jul;3(7):1026–1034. [PubMed]
  • Meerovitch K, Svitkin YV, Lee HS, Lejbkowicz F, Kenan DJ, Chan EK, Agol VI, Keene JD, Sonenberg N. La autoantigen enhances and corrects aberrant translation of poliovirus RNA in reticulocyte lysate. J Virol. 1993 Jul;67(7):3798–3807. [PMC free article] [PubMed]
  • Patton JG, Mayer SA, Tempst P, Nadal-Ginard B. Characterization and molecular cloning of polypyrimidine tract-binding protein: a component of a complex necessary for pre-mRNA splicing. Genes Dev. 1991 Jul;5(7):1237–1251. [PubMed]
  • Hellen CU, Witherell GW, Schmid M, Shin SH, Pestova TV, Gil A, Wimmer E. A cytoplasmic 57-kDa protein that is required for translation of picornavirus RNA by internal ribosomal entry is identical to the nuclear pyrimidine tract-binding protein. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7642–7646. [PMC free article] [PubMed]
  • Hellen CU, Pestova TV, Litterst M, Wimmer E. The cellular polypeptide p57 (pyrimidine tract-binding protein) binds to multiple sites in the poliovirus 5' nontranslated region. J Virol. 1994 Feb;68(2):941–950. [PMC free article] [PubMed]
  • Jang SK, Wimmer E. Cap-independent translation of encephalomyocarditis virus RNA: structural elements of the internal ribosomal entry site and involvement of a cellular 57-kD RNA-binding protein. Genes Dev. 1990 Sep;4(9):1560–1572. [PubMed]
  • Borovjagin AV, Evstafieva AG, Ugarova TYu, Shatsky IN. A factor that specifically binds to the 5'-untranslated region of encephalomyocarditis virus RNA. FEBS Lett. 1990 Feb 26;261(2):237–240. [PubMed]
  • Borman A, Howell MT, Patton JG, Jackson RJ. The involvement of a spliceosome component in internal initiation of human rhinovirus RNA translation. J Gen Virol. 1993 Sep;74(Pt 9):1775–1788. [PubMed]
  • Ali N, Siddiqui A. Interaction of polypyrimidine tract-binding protein with the 5' noncoding region of the hepatitis C virus RNA genome and its functional requirement in internal initiation of translation. J Virol. 1995 Oct;69(10):6367–6375. [PMC free article] [PubMed]
  • Black AC, Luo J, Watanabe C, Chun S, Bakker A, Fraser JK, Morgan JP, Rosenblatt JD. Polypyrimidine tract-binding protein and heterogeneous nuclear ribonucleoprotein A1 bind to human T-cell leukemia virus type 2 RNA regulatory elements. J Virol. 1995 Nov;69(11):6852–6858. [PMC free article] [PubMed]
  • Haller AA, Semler BL. Stem-loop structure synergy in binding cellular proteins to the 5' noncoding region of poliovirus RNA. Virology. 1995 Feb 1;206(2):923–934. [PubMed]
  • Kaminski A, Hunt SL, Patton JG, Jackson RJ. Direct evidence that polypyrimidine tract binding protein (PTB) is essential for internal initiation of translation of encephalomyocarditis virus RNA. RNA. 1995 Nov;1(9):924–938. [PMC free article] [PubMed]
  • Borman AM, Deliat FG, Kean KM. Sequences within the poliovirus internal ribosome entry segment control viral RNA synthesis. EMBO J. 1994 Jul 1;13(13):3149–3157. [PMC free article] [PubMed]
  • Blyn LB, Chen R, Semler BL, Ehrenfeld E. Host cell proteins binding to domain IV of the 5' noncoding region of poliovirus RNA. J Virol. 1995 Jul;69(7):4381–4389. [PMC free article] [PubMed]
  • Metspalu A, Saarma M, Villems R, Ustav M, Lind A. Interaction of 5-S RNA, 5.8-S RNA and tRNA with rat-liver ribosomal proteins. Eur J Biochem. 1978 Nov 2;91(1):73–81. [PubMed]
  • Fahnestock SR, Nomura M. Activity of ribosomes containing 5S RNA with a chemically modified 3'-terminus. Proc Natl Acad Sci U S A. 1972 Feb;69(2):363–365. [PMC free article] [PubMed]
  • Dildine SL, Semler BL. Conservation of RNA-protein interactions among picornaviruses. J Virol. 1992 Jul;66(7):4364–4376. [PMC free article] [PubMed]
  • Luban J, Goff SP. Binding of human immunodeficiency virus type 1 (HIV-1) RNA to recombinant HIV-1 gag polyprotein. J Virol. 1991 Jun;65(6):3203–3212. [PMC free article] [PubMed]
  • Hellman U, Wernstedt C, Góez J, Heldin CH. Improvement of an "In-Gel" digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Anal Biochem. 1995 Jan 1;224(1):451–455. [PubMed]
  • Davis MT, Stahl DC, Hefta SA, Lee TD. A microscale electrospray interface for on-line, capillary liquid chromatography/tandem mass spectrometry of complex peptide mixtures. Anal Chem. 1995 Dec 15;67(24):4549–4556. [PubMed]
  • Leffers H, Dejgaard K, Celis JE. Characterisation of two major cellular poly(rC)-binding human proteins, each containing three K-homologous (KH) domains. Eur J Biochem. 1995 Jun 1;230(2):447–453. [PubMed]
  • Simoes EA, Sarnow P. An RNA hairpin at the extreme 5' end of the poliovirus RNA genome modulates viral translation in human cells. J Virol. 1991 Feb;65(2):913–921. [PMC free article] [PubMed]
  • Aasheim HC, Loukianova T, Deggerdal A, Smeland EB. Tissue specific expression and cDNA structure of a human transcript encoding a nucleic acid binding [oligo(dC)] protein related to the pre-mRNA binding protein K. Nucleic Acids Res. 1994 Mar 25;22(6):959–964. [PMC free article] [PubMed]
  • Wilm M, Shevchenko A, Houthaeve T, Breit S, Schweigerer L, Fotsis T, Mann M. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature. 1996 Feb 1;379(6564):466–469. [PubMed]
  • Patterson SD, Aebersold R. Mass spectrometric approaches for the identification of gel-separated proteins. Electrophoresis. 1995 Oct;16(10):1791–1814. [PubMed]
  • Trono D, Andino R, Baltimore D. An RNA sequence of hundreds of nucleotides at the 5' end of poliovirus RNA is involved in allowing viral protein synthesis. J Virol. 1988 Jul;62(7):2291–2299. [PMC free article] [PubMed]

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