The human insulin receptor mRNA contains a functional internal ribosome entry segment

Spriggs, K. A., Cobbold, L. C., Ridley, S. H., Coldwell, M., Bottley, A., Bushell, M. , Willis, A. E. and Siddle, K. (2009) The human insulin receptor mRNA contains a functional internal ribosome entry segment. Nucleic Acids Research, 37(17), pp. 5881-5893. (doi: 10.1093/nar/gkp623) (PMID:19654240) (PMCID:PMC2761284)

Abstract

Regulation of mRNA translation is an important mechanism determining the level of expression of proteins in eukaryotic cells. Translation is most commonly initiated by cap-dependent scanning, but many eukaryotic mRNAs contain internal ribosome entry segments (IRESs), providing an alternative means of initiation capable of independent regulation. Here, we show by using dicistronic luciferase reporter vectors that the 5′-UTR of the mRNA encoding human insulin receptor (hIR) contains a functional IRES. RNAi-mediated knockdown showed that the protein PTB was required for maximum IRES activity. Electrophoretic mobility shift assays confirmed that PTB1, PTB2 and nPTB, but not unr or PTB4, bound to hIR mRNA, and deletion mapping implicated a CCU motif 448 nt upstream of the initiator AUG in PTB binding. The IR-IRES was functional in a number of cell lines, and most active in cells of neuronal origin, as assessed by luciferase reporter assays. The IRES was more active in confluent than sub-confluent cells, but activity did not change during differentiation of 3T3-L1 fibroblasts to adipocytes. IRES activity was stimulated by insulin in sub-confluent cells. The IRES may function to maintain expression of IR protein in tissues such as the brain where mRNA translation by cap-dependent scanning is less effective.

• Item Type: Articles
• Additional Information: UK Biotechnology and Biological Sciences Research Council (to L.C.C., M.D., A.B., A.E.W., K.S.); Diabetes UK (R.D. Lawrence Fellowship to S.H.R.); European Union COST Action BM0602. Funding for open access charge: Biotechnology and Biological Sciences Research Council grant.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Bushell, Professor Martin
• Authors: Spriggs, K. A., Cobbold, L. C., Ridley, S. H., Coldwell, M., Bottley, A., Bushell, M., Willis, A. E., and Siddle, K.
• College/School: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Journal Name: Nucleic Acids Research
• Publisher: Oxford University Press
• ISSN: 0305-1048
• ISSN (Online): 1362-4962
• Published Online: 04 August 2009
• Copyright Holders: Copyright © 2009 The Authors
• First Published: First published in Nucleic Acids Research 37(17):5881-5893
• Publisher Policy: Reproduced under a Creative Commons license