Mechanical stress and single nucleotide variants regulate alternative splicing of the mylk gene

Joseph B. Mascarenhas, Alex Y. Tchourbanov, Hanli Fan, Sergei M. Danilov, Ting Wang, Joe GN Garcia

Research output: Contribution to journalArticle

5 Citations (Scopus)

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 thenmMLCK2variant lacks exon 11, which encodes a key regulatory region containing two differentially phosphorylated tyrosine residues (Y464 and Y471) 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.

Original languageEnglish (US)
Pages (from-to)29-37
Number of pages9
JournalAmerican Journal of Respiratory Cell and Molecular Biology
Volume56
Issue number1
DOIs
StatePublished - Jan 1 2017
Externally publishedYes

Fingerprint

Mechanical Stress
Alternative Splicing
Polymorphism
Nucleotides
Genes
Single Nucleotide Polymorphism
Endothelial cells
Exons
Endothelial Cells
Blood Vessels
Myosin-Light-Chain Kinase
Nucleic Acid Regulatory Sequences
Computer Simulation
Tyrosine
Protein Isoforms
Cells
Inflammation
Cell Line
Polymerase Chain Reaction
Lung

Keywords

  • Bioinformatics
  • Cyclic stretch
  • Single nucleotide polymorphism
  • Splicing
  • Ventilator-induced lung injury

ASJC Scopus subject areas

  • Medicine(all)
  • Molecular Biology
  • Pulmonary and Respiratory Medicine
  • Clinical Biochemistry
  • Cell Biology

Cite this

Mechanical stress and single nucleotide variants regulate alternative splicing of the mylk gene. / Mascarenhas, Joseph B.; Tchourbanov, Alex Y.; Fan, Hanli; Danilov, Sergei M.; Wang, Ting; Garcia, Joe GN.

In: American Journal of Respiratory Cell and Molecular Biology, Vol. 56, No. 1, 01.01.2017, p. 29-37.

Research output: Contribution to journalArticle

Mascarenhas, Joseph B. ; Tchourbanov, Alex Y. ; Fan, Hanli ; Danilov, Sergei M. ; Wang, Ting ; Garcia, Joe GN. / Mechanical stress and single nucleotide variants regulate alternative splicing of the mylk gene. In: American Journal of Respiratory Cell and Molecular Biology. 2017 ; Vol. 56, No. 1. pp. 29-37.
@article{ffeb9131c0114a69bbbce1184067391a,
title = "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 thenmMLCK2variant lacks exon 11, which encodes a key regulatory region containing two differentially phosphorylated tyrosine residues (Y464 and Y471) 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.",
keywords = "Bioinformatics, Cyclic stretch, Single nucleotide polymorphism, Splicing, Ventilator-induced lung injury",
author = "Mascarenhas, {Joseph B.} and Tchourbanov, {Alex Y.} and Hanli Fan and Danilov, {Sergei M.} and Ting Wang and Garcia, {Joe GN}",
year = "2017",
month = "1",
day = "1",
doi = "10.1165/rcmb.2016-0053OC",
language = "English (US)",
volume = "56",
pages = "29--37",
journal = "American Journal of Respiratory Cell and Molecular Biology",
issn = "1044-1549",
publisher = "American Thoracic Society",
number = "1",

}

TY - JOUR

T1 - Mechanical stress and single nucleotide variants regulate alternative splicing of the mylk gene

AU - Mascarenhas, Joseph B.

AU - Tchourbanov, Alex Y.

AU - Fan, Hanli

AU - Danilov, Sergei M.

AU - Wang, Ting

AU - Garcia, Joe GN

PY - 2017/1/1

Y1 - 2017/1/1

N2 - 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 thenmMLCK2variant lacks exon 11, which encodes a key regulatory region containing two differentially phosphorylated tyrosine residues (Y464 and Y471) 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.

AB - 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 thenmMLCK2variant lacks exon 11, which encodes a key regulatory region containing two differentially phosphorylated tyrosine residues (Y464 and Y471) 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.

KW - Bioinformatics

KW - Cyclic stretch

KW - Single nucleotide polymorphism

KW - Splicing

KW - Ventilator-induced lung injury

UR - http://www.scopus.com/inward/record.url?scp=85008449880&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85008449880&partnerID=8YFLogxK

U2 - 10.1165/rcmb.2016-0053OC

DO - 10.1165/rcmb.2016-0053OC

M3 - Article

VL - 56

SP - 29

EP - 37

JO - American Journal of Respiratory Cell and Molecular Biology

JF - American Journal of Respiratory Cell and Molecular Biology

SN - 1044-1549

IS - 1

ER -