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Members of the poly (rC) binding protein family stimulate the activity of the c-myc internal ribosome entry segment in vitro and in vivo

Oncogene volume 22, pages 8012–8020 (2003)Cite this article

Abstract

The 5′ untranslated region of the proto-oncogene c-myc contains an internal ribosome entry segment and c-Myc translation can be initiated by cap-independent as well as cap-dependent mechanisms. In contrast to the process of cap-dependent initiation, the trans-acting factor requirements for cellular internal ribosome entry are poorly understood. Here, we show that members of the poly (rC) binding protein family, poly (rC) binding protein 1 (PCBP1), poly (rC) binding protein 2 (PCBP2) and hnRNPK were able to activate the IRES in vitro up to threefold when added in combination with upstream of N-ras and unr-interacting protein. The interactions of PCBP1, PCBP2 and hnRNPK with c-myc-IRES-RNA were shown to be specific by ultraviolet crosslinking analysis and electrophoretic mobility shift assays, while immunoprecipitation of the three proteins using specific antibodies followed by reverse transcriptase–polymerase chain reaction showed that they were able to bind c-myc mRNA. c-myc–IRES-mediated translation from the reporter vector was stimulated by cotransfection of plasmids encoding PCBP1, PCBP2 and hnRNPK. Interestingly, the mutated version of the c-myc IRES that is prevalent in patients with multiple myeloma bound hnRNPK more efficiently in vitro and was stimulated by hnRNPK to a greater extent in vivo.

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Acknowledgements

This work was funded by grants from the BBSRC (advanced fellowship held by AEW, project grant funding to KAS) and the Wellcome Trust (SAM). JRE was supported by an MRC studentship. Work in KB laboratory is supported by grants form the NIH and JDRF.

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Authors and Affiliations

  1. Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7RH, UK

    Joanne R Evans, Sally A Mitchell, Keith A Spriggs & Anne E Willis

  2. University of Washington, Seattle, 98195, WA, USA

    Jerzy Ostrowski & Karol Bomsztyk

  3. Department of Biochemistry, Martin-Luther-University, Kurt-Mothes Str. 3, Halle, D-06210, Germany

    Dirk Ostarek

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  1. Joanne R Evans
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  2. Sally A Mitchell
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  7. Anne E Willis
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Correspondence to Anne E Willis.

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Evans, J., Mitchell, S., Spriggs, K. et al. Members of the poly (rC) binding protein family stimulate the activity of the c-myc internal ribosome entry segment in vitro and in vivo. Oncogene 22, 8012–8020 (2003). https://doi.org/10.1038/sj.onc.1206645

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