Initiation of translation from most cellular mRNAs occurs via scanning; the 40 S ribosomal subunit binds to the m(7)G-cap and then moves along the
mRNA until an
initiation codon is encountered. Some cellular mRNAs contain internal ribosome entry sequences (IRESs) within their 5'-untranslated regions, which allow initiation independently of the 5'-cap. This study investigated the ability of cellular stress to regulate the activity of IRESs in cellular mRNAs. Three stresses were studied that cause the phosphorylation of the translation
initiation factor, eIF2alpha, by activating specific
kinases: (i)
amino acid starvation, which activates GCN2; (ii) endoplasmic reticulum (ER) stress, which activates PKR-like ER
kinase,
PERK kinase; and (iii)
double-stranded RNA, which activates
double-stranded RNA-dependent
protein kinase (PKR) by mimicking
viral infection.
Amino acid starvation and ER stress caused transient phosphorylation of eIF2alpha during the first hour of treatment, whereas
double-stranded RNA caused a sustained phosphorylation of eIF2alpha after 2 h. The effects of these treatments on IRES-mediated initiation were investigated using bicistronic
mRNA expression vectors. No effect was seen for the IRESs from the mRNAs for the chaperone BiP and the
protein kinase Pim-1. In contrast, translation mediated by the IRESs from the
cationic amino acid transporter, cat-1, and of the cricket paralysis virus
intergenic region, were stimulated 3- to 10-fold by all three treatments. eIF2alpha phosphorylation was required for the response because inactivation of phosphorylation prevented the stimulation. It is concluded that cellular stress can stimulate translation from some cellular IRESs via a mechanism that requires the phosphorylation of eIF2alpha. Moreover, there are distinct regulatory patterns for different cellular mRNAs that contain IRESs within their 5'-untranslated regions.