Alternative mRNA Splicing Analysis of DAF-2 During Hydrogen Peroxide Stress

Abstract

Alternative mRNA splicing is a mechanism of regulating gene expression accomplished by varying which protein-coding sequences are included in the mature mRNA. It is known that various types of stress influence alternative splicing. Thus, the concentration, variety, and functionality of proteins produced in a cell and organism. DAF-2 is a gene whose protein functions in the insulin pathway in many organisms including the nematode, C. elegans. This pathway is known to be altered during the stress response. Our hypothesis was that nematodes will alternatively splice DAF-2 mRNA in the region encoding the tyrosine kinase domain (exons 10-13) upon exposure to the environmental stressor, hydrogen peroxide. To test this hypothesis, nematodes were grown under standard conditions, isolated, and exposed to either 20 mM H2O2 or diluent only. RNA was isolated and analyzed by reverse transcription-polymerase chain reaction. The results were visualized following agarose gel electrophoresis. Four distinct and specific cDNA fragments were observed in the control sample indicating that the mRNA had been spliced in four different ways. Only one of these distinct and specific cDNA fragments was observed in stressed worms. This clearly indicated that alternative splicing had occurred due to the peroxide stress. The largest cDNA fragment is the size expected if all exons were included and thus the tyrosine kinase domain would be encoded. The smallest cDNA fragment is the size expected if exons 11 and 12 were skipped and thus the tyrosine kinase domain would be absent from the DAF-2 protein. The two middle size fragments were not predicted. These may represent skipping of exons 11 and 12 individually. Cloning and sequencing of the fragments should be done to confirm the identity of these assumptions. In addition, the experiment should also be performed with different peroxide concentrations and for varying times to analyze the kinetics and timing of alternative splicing in response to this stressor.