Post‐transcriptional modulation of aENaC mRNA stability in alveolar epithelial cells: involvement of conserved domains on its 3′ untranslated region
Bibliographic record
Abstract
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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".