Mechanical Stress and Single Nucleotide Variants Regulate Alternative Splicing of the MYLK Gene

Abstract

The nonmuscle (nm) myosin light-chain kinase isoform (MLCK), encoded by the MYLK gene, is a vital participant in regulating vascular barrier responses to mechanical and inflammatory stimuli. We determined that MYLK is alternatively spliced, yielding functionally distinct nmMLCK splice variants including nmMLCK2, a splice variant highly expressed in vascular endothelial cells (EC) and associated with reduced EC barrier integrity. We demonstrated previously that the nmMLCK2 variant lacks exon 11, which encodes a key regulatory region containing two differentially phosphorylated tyrosine residues (Y and Y) that influence vascular barrier function during inflammation. In this study, we used minigene constructs and RT-PCR to interrogate biophysical factors (mechanical stress) and genetic variants (MYLK single-nucleotide polymorphisms [SNPs]) that are potentially involved in regulating MYLK alternative splicing and nmMLCK2 generation. Human lung EC exposed to pathologic mechanical stress (18% cyclic stretch) produced increased nmMLCK2 expression relative to levels of nmMLCK1 with alternative splicing significantly influenced by MYLK SNPs rs77323602 and rs147245669. In silico analyses predicted that these variants would alter exon 11 donor and acceptor sites for alternative splicing, computational predictions that were confirmed by minigene studies. The introduction of rs77323602 favored wild-type nmMLCK expression, whereas rs147245669 favored alternative splicing and deletion of exon 11, yielding increased nmMLCK2 expression. Finally, lymphoblastoid cell lines selectively harboring these MYLK SNPs (rs77323602 and rs147245669) directly validated SNP-specific effects on MYLK alternative splicing and nmMLCK2 generation. Together, these studies demonstrate that mechanical stress and MYLK SNPs regulate MYLK alternative splicing and generation of a splice variant, nmMLCK2, that contributes to the severity of inflammatory injury.

The Splicing Factor hnRNPA1 Regulates Alternate Splicing of the MYLK Gene

“…We recently used a minigene approach to address mechanisms that potentially underlie the regulation of MYLK splicing (15) and demonstrated the functional role of SNPs in altering MYLK splicing (15). It is well known that pre-mRNA splicing occurs through the activity of the spliceosome comprised of the heterogeneous nuclear ribonucleoproteins (hnRNPs), small nuclear ribonucleoprotein particles, and other components of the splicing machinery (16).…”

“…The minigene construct was constructed as we recently reported (15). Site-directed mutagenesis was performed using the QuikChange II site-directed mutagenesis kit (Agilent Technologies).…”

“…Constructs were transfected into human embryonic kidney 293 (HEK-293) cells. RNA isolation and RT were performed as described elsewhere (15). PCR and quantitation of products were performed as described above.…”

Genome-Wide Association Study in African Americans with Acute Respiratory Distress Syndrome Identifies the Selectin P Ligand Gene as a Risk Factor

“…Racial and ethnic differences in susceptibility to and mortality from ARDS (12)(13)(14)(15)(16) support the increasingly recognized role of genetic factors in ARDS susceptibility (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27), and highlight the importance of identifying genetic biomarkers of ARDS susceptibility in atrisk individuals to further improve risk stratification (28). We have previously identified several novel genetic ARDS biomarkers and therapeutic targets, including NAMPT (nicotinamide phosphoribosyl transferase), which encodes for a Toll-like receptor 4-binding cytozyme (17,24), MYLK (encoding MLCK [myosin lightchain kinase]) (19,26,29), GADD45a (growth arrest and DNA damage-inducible gene) (30), and sphingosine 1-phosphate receptor 3 (25).…”

Myosin light chain kinase (MYLK) coding polymorphisms modulate human lung endothelial cell barrier responses via altered tyrosine phosphorylation, spatial localization, and lamellipodial protrusions

“…c-Src or c-Abl) compared to nmMLCK2 which has exon 11 deleted via alternative splicing. 1 , 32 We recently demonstrated that ARDS-related stimuli drive alternative splicing that favors greater expression of nmMLCK2, and thus less regulation by barrier-restoring stimuli, such as S1P and HGF, and consequently persistent gap formation. 32…”