The Splicing Factor hnRNPA1 Regulates Alternate Splicing of the MYLK Gene.

Abstract

Profound lung vascular permeability is a cardinal feature of acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI), two syndromes known to centrally involve the nonmuscle isoform of myosin light chain kinase (nmMLCK) in vascular barrier dysregulation. Two main splice variants, nmMLCK1 and nmMLCK2, are well represented in human lung endothelial cells and encoded by MYLK, and they differ only in the presence of exon 11 in nmMLCK1, which contains critical phosphorylation sites (Y464 and Y471) that influence nmMLCK enzymatic activity, cellular translocation, and localization in response to vascular agonists. We recently demonstrated the functional role of SNPs in altering MYLK splicing, and in the present study we sought to identify the role of splicing factors in the generation of nmMLCK1 and nmMLCK2 spliced variants. Using bioinformatic in silico approaches, we identified a putative binding site for heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), a recognized splicing factor. We verified hnRNPA1 binding to MYLK by gel shift analyses and that hnRNPA1 gene and protein expression is upregulated in mouse lungs obtained from preclinical models of ARDS and VILI and in human endothelial cells exposed to 18% cyclic stretch, a model that reproduces the excessive mechanical stress observed in VILI. Using an MYLK minigene approach, we established a direct role of hnRNPA1 in MYLK splicing and in the context of 18% cyclic stretch. In summary, these data indicate an important regulatory role for hnRNPA1 in MYLK splicing, and they increase understanding of MYLK splicing in the regulation of lung vascular integrity during acute lung inflammation and excessive mechanical stress, such as that observed in ARDS and VILI.

• Authors: Joseph B Mascarenhas, Alex Y Tchourbanov, Sergei M Danilov, Tong Zhou, Ting Wang, Joe G N Garcia
• Journal: American journal of respiratory cell and molecular biology (Am J Respir Cell Mol Biol) Vol. 58 Issue 5 Pg. 604-613 (05 2018) ISSN: 1535-4989 [Electronic] United States
• PMID: 29077485 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• Calcium-Binding Proteins
• Heterogeneous Nuclear Ribonucleoprotein A1
• Hnrnpa1 protein, mouse
• MYLK protein, mouse
• hnRNPA1 protein, human
• MYLK protein, human
• Myosin-Light-Chain Kinase
• cardiac myosin light chain kinase, human

Topics

• Alternative Splicing
• Animals
• Binding Sites
• Calcium-Binding Proteins (genetics, metabolism)
• Capillary Permeability
• Disease Models, Animal
• Electric Impedance
• Endothelial Cells (enzymology)
• Exons
• HEK293 Cells
• Heterogeneous Nuclear Ribonucleoprotein A1 (genetics, metabolism)
• Humans
• Introns
• Lung (blood supply)
• Mechanotransduction, Cellular
• Mice
• Myosin-Light-Chain Kinase (genetics, metabolism)
• Protein Binding
• Pulmonary Stretch Receptors (metabolism)
• Respiratory Distress Syndrome (enzymology, genetics, physiopathology)
• Ventilator-Induced Lung Injury (enzymology, genetics, physiopathology)