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Post‐transcriptional modulation of aENaC mRNA stability in alveolar epithelial cells: involvement of conserved domains on its 3′ untranslated region
Author(s) -
Gag Frédéric,
Migneault Francis,
Berthiaume Yves,
Dagenais André
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.596.10
Subject(s) - epithelial sodium channel , untranslated region , messenger rna , three prime untranslated region , microbiology and biotechnology , complementary dna , biology , transfection , mutant , gene , chemistry , genetics , sodium , organic chemistry
The epithelial sodium channel (ENaC) expressed in alveolar epithelial cells (AEC) plays a major role for lung liquid clearance at birth and lung edema resorption in adulthood. We showed previously that different conditions affecting alveolar epithelial cells induce the rapid breakdown of αENaC mRNA by post‐transcriptional modulation of its stability. Since the 3′ untranslated region (3′UTR) of mRNA are known to modulate transcript stability, a sequence analysis was performed comparing αENaC 3′UTR of different species to find conserved homologous domains. Five highly conserved sequences (CS) were identified in the 3′UTR portion of αENaC mRNA, suggesting a role for these sequences in modulation of its stability. Rat αENaC cDNA with full 3′UTR sequences were cloned in pTRE‐tight vector of the TET‐off system. After transfection of AEC by electroporation, transcription of the αENaC TET‐off clone was inhibited with doxycycline. The level of transcript remaining after different time points was evaluated by RT‐PCR. Using this technique, the half‐life (T 1/2 ) of the transcript was evaluated to be ~100min. To study the impact of αENaC 3′UTR sequences and the role of the different CS in modulation of αENaC mRNA stability, 3′UTR deletion mutants were designed by gradually removing the 3′ distal portion of the 3′UTR sequence deleting gradually CS1 (263bp), CS1–2 (454bp), CS1–3 (515bp) and CS1–5 (complete 3′UTR; 894bp). All the deletion mutants affected αENaC mRNA stability. The resulting T 1/2 were respectively of 203 min (CS1), 300 min (CS1–2), 48 min (CS1–3) and 375 min (no 3′UTR). These results suggest that there is a complex stabilizing and destabilizing interplay between cis‐elements present in different regions of 3′UTR to modulate αENaC mRNA stability. Because large portions of 3′UTR were removed in these mutants, deletion mutants targeted to remove specifically the different CS were tested to assess the role of each of these domains in transcript stability. A deletion mutant removing only CS1 (31 bp) shows the same T 1/2 than the first sequential deletion mutant (T 1/2 202 min). The CS2 and CS3 domains are present in close tandem. This is the same for CS4–CS5. Deleting CS2–3 (82 bp) increases T 1/2 to 176 min while deletion of CS4–5 (74 bp) increases the stability of the transcript to 220 min. Altogether, our results show that CS play an important role for destabilization of αENaC mRNA since their removal increases αENaC mRNA T 1/2 . These elements could play an important role in the rapid breakdown of αENaC mRNA during inflammation and injury in the lung.