Abstract
In this report, we demonstrate that cellular stress regulates expression of IFRD1 by a post-transcriptional control mechanism. IFRD1 mRNA and protein are elevated in tunicamycin-treated human kidney epithelial cells via stabilization of the mRNA. IFRD1 mRNA instability in resting cells requires translation of an upstream open reading frame (ORF) that represses translation of the major ORF. During stress response, the mRNA is stabilized via inhibition of translational initiation mediated by phosphorylated eIF2alpha. Translation of the major ORF of IFRD1 involves both leaky scanning at the upstream AUG codon and re-initiation at the major AUG codon and is not altered during stress. Finally, the instability mechanism depends upon UPF1, suggesting that it is related to nonsense-mediated decay. Importantly, the sequence and length of the upstream ORF are critical but do not need to code for a specific peptide. Moreover the sequence environment of the upstream ORF termination site is not an essential feature of instability. These features of decay collectively define a distinct upstream ORF-mediated instability mechanism whereby cellular stress can modulate specific gene expression through alteration of mRNA half-life.
Highlights
Grant CA62220 from USPHS. □S The on-line version of this article contains supplemental Figs. 1–3. 1 To whom correspondence should be addressed: Dept. of Immunology, traumatic injury and infectious challenge are known to initiate inflammatory response, it is becoming increasingly evident that cellular stress mechanisms such as the unfolded protein response (UPR)2 are commonly engaged at sites of tissue injury (14 –16)
To determine if Tm treatment was acting to alter the half-life of IFRD1 mRNA, normal kidney epithelial (NKE) cells were treated with DMSO or Tm for 6 h and further transcription was blocked by the addition of actinomycin D prior to determination of residual IFRD1 transcript 1 (TR1) and GAPDH mRNA levels by real-time RT-PCR (Fig. 1D)
The IFRD1 gene product has been reported to function in cell development or differentiation, its expression has been reported to be consistently elevated in numerous settings involving tissue injury, conditions that may be linked with accompanying cellular stress (10 –13)
Summary
Grant CA62220 from USPHS. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–3. 1 To whom correspondence should be addressed: Dept. of Immunology, traumatic injury and infectious challenge are known to initiate inflammatory response, it is becoming increasingly evident that cellular stress mechanisms such as the unfolded protein response (UPR)2 are commonly engaged at sites of tissue injury (14 –16). The levels of reporter mRNA and protein derived from 5ЈTR1-KC were selectively increased in cells treated with Tm compared with those derived from the KC RBG construct or plasmids containing the full coding region or 3Ј-UTR of human IFRD1.
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