Migneault Francis, Gagnon Frédéric, Pascariu Mihai, Laperle Jonathan, Roy Antoine, Dagenais André, Berthiaume Yves
Institut de Recherches Cliniques de Montréal (IRCM), Montréal, QC, Canada.
Département de Médecine, Faculté de Médecine, Université de Montréal, Montréal, QC, Canada,
Cell Physiol Biochem. 2019;52(5):984-1002. doi: 10.33594/000000068.
BACKGROUND/AIMS: The epithelial sodium channel (ENaC) expressed in alveolar epithelial cells plays a major role in lung liquid clearance at birth and lung edema resorption in adulthood. We showed previously that αENaC mRNA expression is downregulated in part via posttranscriptional regulation of mRNA stability. In the present work, the role of the αENaC 3' untranslated region (3'UTR) in the regulation of mRNA stability was studied further.
Quantitative reverse transcription PCR (qRT-PCR) was performed to investigate the expression of αENaC in alveolar epithelial cells. The role of the αENaC 3'UTR was evaluated through sequential deletions. RNA affinity chromatography and mass spectrometry were achieved to investigate the nature of the proteins that could bind this sequence. The function of these proteins was assessed through knockdown and overexpression in vitro.
First, we found that αENaC mRNA half-life was much shorter than expected when using a transcriptionally controlled plasmid expression system compared to Actinomycin D treatment. Sequential deletions of the αENaC 3'UTR revealed that the αENaC 3'UTR plays an important role in the modulation of αENaC mRNA stability, and that there is a complex stabilizing and destabilizing interplay between different regions of the 3'UTR that modulate this process. Finally, we identified RNA-binding proteins that interact with the αENaC 3'UTR and showed that Dhx36 and Tial1 are involved in the decrease in αENaC mRNA stability via the proximal region of its 3'UTR.
Taken together, these findings indicate that the αENaC 3'UTR plays an important role in modulating transcript levels, and Dhx36 and Tial1 seem to be involved in posttranscriptional regulation of αENaC expression in alveolar epithelial cells.
背景/目的：肺泡上皮细胞中表达的上皮钠通道（ENaC）在出生时肺液清除及成年期肺水肿吸收过程中起主要作用。我们之前表明，αENaC mRNA表达部分通过mRNA稳定性的转录后调控而下调。在本研究中，进一步探究了αENaC 3'非翻译区（3'UTR）在mRNA稳定性调控中的作用。
采用定量逆转录PCR（qRT-PCR）检测肺泡上皮细胞中αENaC的表达。通过连续缺失评估αENaC 3'UTR的作用。采用RNA亲和层析和质谱分析来研究可结合该序列的蛋白质的性质。通过体外敲低和过表达评估这些蛋白质的功能。
首先，我们发现与放线菌素D处理相比，使用转录控制的质粒表达系统时，αENaC mRNA半衰期比预期短得多。αENaC 3'UTR的连续缺失表明，αENaC 3'UTR在调节αENaC mRNA稳定性中起重要作用，并且3'UTR不同区域之间存在复杂的稳定和不稳定相互作用来调节这一过程。最后，我们鉴定了与αENaC 3'UTR相互作用的RNA结合蛋白，并表明Dhx36和Tial1通过其3'UTR的近端区域参与αENaC mRNA稳定性的降低。
综上所述，这些发现表明αENaC 3'UTR在调节转录水平中起重要作用，并且Dhx36和Tial1似乎参与肺泡上皮细胞中αENaC表达的转录后调控。
