Nucleic Acids Research Advance Access originally published online on July 7, 2007
Nucleic Acids Research 2007 35(14):4767-4778; doi:10.1093/nar/gkm512
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Nucleic Acids Research, 2007, Vol. 35, No. 14 4767-4778
© 2007 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.
Molecular Biology |
Cap-independent translation through the p27 5'-UTR
1Cancer Biology Research Institute, Sanford Research/USD, 1400 West 22nd Street, Sioux Falls, South Dakota 57105 and 2Sanford School of Medicine of the University of South Dakota, Division of Basic Biomedical Sciences, 414 East Clark Street, Vermillion, South Dakota 57069, USA
*To whom correspondence should be addressed. Tel: +1 605 357 1544; Fax: +1 605 357 1409; Email: keith.miskimins{at}usd.edu
Received April 4, 2007. Revised June 14, 2007. Accepted June 14, 2007.
Several recent publications have explored cap-independent translation through an internal ribosome entry site (IRES) in the 5'-UTR of the mRNA encoding the cyclin-dependent kinase inhibitor p27. The major experimental tool used in these reports was the use of bicistronic reporter constructs in which the 5'-UTR was inserted between the upstream and downstream cistrons. None of these reports has completely ruled out the possibility that the 5'-UTR has either cryptic promoter activity or a cryptic splice acceptor site. Either of these possibilities could result in expression of a monocistronic mRNA encoding the downstream cistron and false identification of an IRES. Indeed, Liu et al. recently published data suggesting that the p27 5'-UTR harbors cryptic promoter activity which accounts for its putative IRES activity. In this report, we have explored this potential problem further using promoterless bicistronic constructs coupled with RNase protection assays, siRNA knockdown of individual cistrons, RT-PCR to detect mRNA encoded by the bicistronic reporter with high sensitivity, direct transfection of bicistronic mRNAs, and insertion of an iron response element into the bicistronic reporter. The results do not support the conclusion that the p27 5'-UTR has significant functional promoter activity or cryptic splice sites, but rather that it is able to support cap-independent initiation of translation.
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