S. F. Monaghan1, K. J. Cygan2, J. Lomas-Neira1, C. Chun1, Y. Chen1, W. G. Cioffi1, W. G. Fairbrother2, A. Ayala1 1Brown University School Of Medicine,Surgery,Providence, RI, USA 2Brown University,Center For Computational Molecular Biology,Providence, RI, USA
Introduction: Previous work has focused on the regulation of transcription of DNA into RNA as it relates to critical illness. However, the next step in protein production, RNA splicing, has not received much attention. Over 90% of human genes with multiple exons have alternative splicing events. With such a high rate of variations from the transcribed gene to the protein, splicing must be under exquisite control; particularly when cellular resources are limited during critical illness. Here we hypothesize RNA splicing is altered in critical illness as evidenced by changes in the gene expression of RNA splicing protein.
Methods: A murine model of acute respiratory distress syndrome (ARDS) was induced by subjecting mice to hemorrhagic shock followed by cecal ligation and puncture. Mice undergoing severe critical illness from this model were compared to sham controls. Blood and lung samples were collected and RNA was purified from the samples using the Globinclear (Ambion) and MasterPure RNA purification (Epicentre) kits. Next generation RNA sequencing was performed (Genewiz). Analysis of the RNA sequencing data was done using the STAR aligner and the GenomicAlignments package for R/Bioconductor. Genes were categorized as RNA splicing proteins using gene ontology (GO) terms. Alpha was set at 0.05 while adjusting for the number reads obtained.
Results:Gene expression in mice with induced critical illness (ARDS) were compared to sham controls. More genes in the blood (17%) had significantly different expression levels between the groups (critically ill mouse vs sham control) when compared to the genes in the lung tissue (4%, p=0.0001). 618 genes had significant changes in expression in both the blood and lung tissue. Using GO terms, 17 RNA splicing genes were significantly changed in the blood and only one gene was identified in the lung (see table, in order of amount of change). Of the 17 genes with significantly different expression 5 showed an increased in expression while 12 showed a decrease in expression. Although fewer genes identified had more expression, they had a cumulative 86 fold change in expression compared to a 46 fold change in expression of the 12 genes that decreased. The one gene identified in the lung showed small decrease in expression.
Conclusion:Numerous genes involved in RNA splicing show significant changes in expression, particularly in the blood, in an animal model system of critical illness. Since resources are limited in critical illness, the alterations in the expression of these RNA splicing proteins suggests there will be a change in the RNA processing during times of stress. Future work will assess changes in alternative splicing in critical illness and attempt to understand this process.
