Nucleic Acids Research Advance Access originally published online on August 4, 2009
Nucleic Acids Research 2009 37(17):5881-5893; doi:10.1093/nar/gkp623
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Nucleic Acids Research, 2009, Vol. 37, No. 17 5881-5893
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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The human insulin receptor mRNA contains a functional internal ribosome entry segment
1University of Nottingham, School of Pharmacy, University Park, Nottingham NG7 2RD, 2University of Cambridge, Department of Clinical Biochemistry, Metabolic Research Laboratories, Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge CB2 0QQ and 3School of Biological Sciences, University of Southampton, Boldrewood Campus, Bassett Crescent East, Southampton, SO16 7PX, UK
*To whom correspondence should be addressed. Tel: 44 1223 336789; Fax: 44 1223 330598; Email: ks14{at}mole.bio.cam.ac.uk
Correspondence may also be addressed to Anne E. Willis. Tel: 44 115 8467095; Fax: 44 115 8468877; Email: anne.willis{at}nottingham.ac.uk
Received March 30, 2009. Revised June 18, 2009. Accepted July 11, 2009.
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.