Dataset Information

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 (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.

SUBMITTER: Mascarenhas JB

PROVIDER: S-EPMC5248959 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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

Mascarenhas Joseph B JB Tchourbanov Alex Y AY Fan Hanli H Danilov Sergei M SM Wang Ting T Garcia Joe G N JG

American journal of respiratory cell and molecular biology 20170101 1

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, ...[more]

PMID: 27529643

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Project description:It is estimated that more than 90% of human genes express multiple mRNA transcripts due to alternative splicing. Consequently, the proteins produced by different splice variants will likely have different functions and expression levels. Several genes with splice variants are known in bone, with functions that affect osteoblast function and bone formation. The primary goal of this study was to evaluate the extent of alternative splicing in a bone subjected to mechanical loading and subsequent bone formation. We used the rat forelimb loading model, in which the right forelimb was loaded axially for 3 min, while the left forearm served as a non-loaded control. Animals were subjected to loading sessions every day, with 24 h between sessions. Ulnae were sampled at 11 time points, from 4 h to 32days after beginning loading. RNA was isolated and mRNA abundance was measured at each time point using Affymetrix exon arrays (GeneChip® Rat Exon 1.0 ST Arrays). An ANOVA model was used to identify potential alternatively spliced genes across the time course, and five alternatively spliced genes were validated with qPCR: Akap12, Fn1, Pcolce, Sfrp4, and Tpm1. The number of alternatively spliced genes varied with time, ranging from a low of 68 at 12h to a high of 992 at 16d. We identified genes across the time course that encoded proteins with known functions in bone formation, including collagens, matrix proteins, and components of the Wnt/?-catenin and TGF-? signaling pathways. We also identified alternatively spliced genes encoding cytokines, ion channels, muscle-related genes, and solute carriers that do not have a known function in bone formation and represent potentially novel findings. In addition, a functional characterization was performed to categorize the global functions of the alternatively spliced genes in our data set. In conclusion, mechanical loading induces alternative splicing in bone, which may play an important role in the response of bone to mechanical loading.

Dataset Information

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

Publications

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

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