Email updates

Keep up to date with the latest news and content from BMC Molecular Biology and BioMed Central.

Open Access Methodology article

Transcripts expressed using a bicistronic vector pIREShyg2 are sensitized to nonsense-mediated mRNA decay

Yayoi Shikama1*, Huiyuan Hu12, Makiko Ohno1, Isao Matsuoka13, Tsutomu Shichishima45 and Junko Kimura1

Author Affiliations

1 Department of Pharmacology, Fukushima Medical University, Fukushima, Japan

2 Department of Pharmaceutical Toxicology, School of Pharmaceutical Sciences, China Medical University, Shenyang, China

3 The Laboratory of Pharmacology, Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki, Japan

4 Department of Cardiology and Hematology, Fukushima Medical University, Fukushima, Japan

5 Fukushima Research Institute of Environment and Medicine, Futaba, Japan

For all author emails, please log on.

BMC Molecular Biology 2010, 11:42  doi:10.1186/1471-2199-11-42

Published: 1 June 2010

Abstract

Background

pIREShyg2 has been widely used as a bicistronic expression vector. However, it is not known if the vector would affect the expression of cloned genes via nonsense-mediated mRNA decay (NMD), an mRNA surveillance system that degrades mRNA with a premature termination codon (PTC). In mammalian cells, the induction of NMD requires either a long 3'UTR or the presence of an exon-junction complex downstream of a PTC. The efficiency of NMD is greater when a PTC generates longer 3'UTR. pIREShyg2 provides the first cistron gene with a long 3'UTR consisting of a downstream intervening sequence (IVS), an internal ribosomal entry site (IRES) and the second cistron. Therefore, we hypothesized that the first cistron genes in pIREShyg2 are sensitized to NMD, which affects their expression levels. To examine this hypothesis, cDNAs encoding human granulocyte-macrophage colony-stimulating factor receptor β chain (βc) and its splice variant (βc79), in which the retention of a 79-base intron caused a frameshift generating 18 PTCs, were cloned into pIREShyg2 and stably expressed in a murine cell line, Ba/F3.

Results

Compared with wild-type βc, the mRNA levels of βc79 were less than one tenth and decayed faster. Both translation inhibition and Upf1 knockdown led to significantly greater up-regulation of βc79 than wild-type βc. However, the use of a monocistronic pMT21 vector abolished the up-regulatory effects of translation inhibition and Upf1 knockdown on both wild-type βc and βc79, suggesting that the NMD is attributable to a structural determinant in pIREShyg2. The elimination of the intron and the proximal 3' 17 PTCs did not alter the greater effects of translation inhibition on βc79, suggesting that the first PTC, which determines 3'UTR length, was sufficient to enhance NMD efficiency. Thus, transcripts of PTC-harboring genes with longer 3'UTR are more efficiently degraded by the vector-dependent NMD than those of wild-type genes with relatively shorter 3'UTR, resulting in minimized expression of truncated mutants.

Conclusions

We conclude that pIREShyg2, which sensitizes its bicistronic transcripts to NMD, may be useful for studying NMD but should be avoided when maximum expressions of PTC-harboring genes are required.