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


RESULTS
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.


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.
Shikama
Yayoi
Y
Department of Pharmacology, Fukushima Medical University, Fukushima, Japan. yayois@fmu.ac.jp
Hu
Huiyuan
H
Ohno
Makiko
M
Matsuoka
Isao
I
Shichishima
Tsutomu
T
Kimura
Junko
J
D020413:3' Untranslated Regions; D017398:Alternative Splicing; D002460:Cell Line; D018389:Codon, Nonsense; D016368:Frameshift Mutation; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007438:Introns; D020365:RNA Helicases; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism*; D015534:Trans-Activators / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation
D020413:3' Untranslated Regions; D018389:Codon, Nonsense; D012333:RNA, Messenger; D015534:Trans-Activators; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D020365:RNA Helicases; C101828:UPF1 protein, human