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

ABSTRACT: 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 beta chain (betac) and its splice variant (betac79), 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.Compared with wild-type betac, the mRNA levels of betac79 were less than one tenth and decayed faster. Both translation inhibition and Upf1 knockdown led to significantly greater up-regulation of betac79 than wild-type betac. However, the use of a monocistronic pMT21 vector abolished the up-regulatory effects of translation inhibition and Upf1 knockdown on both wild-type betac and betac79, 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 betac79, 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.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.

SUBMITTER: Shikama Y

PROVIDER: S-EPMC2896932 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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

Shikama Yayoi Y Hu Huiyuan H Ohno Makiko M Matsuoka Isao I Shichishima Tsutomu T Kimura Junko J

BMC molecular biology 20100601

<h4>Background</h4>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. ...[more]

PMID: 20513249

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Project description:Eukaryotic polycistronic transcription units are rare and only a few examples are known, mostly being the outcome of serendipitous discovery. We claim that nonsense-mediated mRNA decay (NMD) immune structure is a common characteristic of polycistronic transcripts, and that this immunity is an emergent property derived from all functional CDSs. The human RefSeq transcriptome was computationally screened for transcripts capable of eliciting NMD, and which contain an additional ORF(s) potentially capable of rescuing the transcript from NMD. Transcripts were further analyzed implementing domain-based strategies in order to estimate the potential of the candidate ORF to encode a functional protein. Consequently, we predict the existence of forty nine novel polycistronic transcripts. Experimental verification was carried out utilizing two different types of analyses. First, five Gene Expression Omnibus (GEO) datasets from published NMD-inhibition studies were used, aiming to explore whether a given mRNA is indeed insensitive to NMD. All known bicistronic transcripts and eleven out of the twelve predicted genes that were analyzed, displayed NMD insensitivity using various NMD inhibitors. For three genes, a mixed expression pattern was observed presenting both NMD sensitivity and insensitivity in different cell types. Second, we used published global translation initiation sequencing data from HEK293 cells to verify the existence of translation initiation sites in our predicted polycistronic genes. In five of our genes, the predicted rescuing uORFs are indeed identified as translation initiation sites, and in two additional genes, one of two predicted rescuing uORF is verified. These results validate our computational analysis and reinforce the possibility that NMD-immune architecture is a parameter by which polycistronic genes can be identified. Moreover, we present evidence for NMD-mediated regulation controlling the production of one or more proteins encoded in the polycistronic transcript.

Dataset Information

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

Publications

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

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