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

Area of Science:

• Molecular Biology
• Gene Expression Regulation
• mRNA Surveillance

Background:

• The pIREShyg2 vector is a commonly used bicistronic expression system.
• The impact of pIREShyg2 on nonsense-mediated mRNA decay (NMD) was previously unknown.
• NMD degrades mRNA containing premature termination codons (PTCs), influenced by 3' untranslated region (3'UTR) length and exon-junction complexes.

Purpose of the Study:

• To investigate whether the pIREShyg2 vector sensitizes genes to NMD.
• To determine if pIREShyg2 affects the expression levels of cloned genes via NMD.
• To test the hypothesis that the first cistron in pIREShyg2 is susceptible to NMD.

Main Methods:

• Cloned cDNAs for human granulocyte-macrophage colony-stimulating factor receptor beta chain (betac) and a splice variant (betac79) into pIREShyg2.
• Expressed constructs in a murine Ba/F3 cell line.
• Analyzed mRNA levels, decay rates, and effects of translation inhibition and Upf1 knockdown.

Main Results:

• betac79 mRNA levels were significantly lower and decayed faster than wild-type betac.
• Translation inhibition and Upf1 knockdown caused greater upregulation of betac79 compared to betac.
• Vector-dependent NMD in pIREShyg2 was confirmed, with longer 3'UTRs from PTCs enhancing degradation.

Conclusions:

• The pIREShyg2 vector sensitizes bicistronic transcripts to NMD.
• This vector-mediated NMD leads to reduced expression of genes with PTCs.
• pIREShyg2 is suitable for NMD studies but not for maximizing expression of PTC-containing genes.