Study The Regulation Of Alternative Splicing In The Hypoxic Response By C. Elegans As A Model Organism

Oxygen has an important role in aerobic organisms, lacking oxygen will cause injury or hypoxia-related diseases in the body. In order to adapt to hypoxic stress, organisms need to regulate transcription and expression of gene hypoxia-related. Alternative splicing is an important way that regulates the gene expression, and the main reason for the complexity of proteome and gene expression patterns in eukaryotic. Studies have reported that many gene hypoxia-related involve in hypoxia response in different splice variants by alternative splicing and play an important role. Alternative splicing closely related to the pathogenesis of many diseases hypoxia-related, but the specific regulatory mechanism that alternative splicing in the hypoxia response is poorly understood.To investigate the regulation of alternative splicing in hypoxia response, in this topic, we make the model organism C. elegans as the material, using feeding RNAi screening technology, with 351 RNA binding protein (RNA-binding protein, RBP) coding genes and the the gene that having experimental evidence to support that have alternative splicing forms(candidate target genes) in C. elegans for the screening object, to find the splicing regulatory factors and target genes that affect the hypoxic injury phenotype of C. elegans. The results showed hypoxia phenotype have significant differences after 10 genes RNAi compared with N2, including 6 RBP genes (F46A9.6, T26G10.1, M03D4.6, R09A1.1, T07D1. 4, F18H3.3), and 4 candidate target genes (W01C9.5, H21P03.2, F22E12.4, K09G1.4). Because RNAi has some uncertainty, so we need to use loss of function mutants of positive gene for further validation. The results showed that the physical hypoxic phenotype of mec-8(ok2043), mec-8(u314) and egl-9(sa307) mutants consistent with RNAi, in which hypoxic phenotype of mec-8(ok2043) are hypoxia-sensitive phenotype, while the hypoxic phenotype of mec-8(u314) and egl-9(sa307) are hypoxia-tolerance phenotype. The data suggest that genes mec-8 and egl-9 may play an important role in response to hypoxia. To further prove that egl-9 involve in hypoxia response at the molecular level, and which variations involve in hypoxia response, we assay the mRNA expression levels of some splice variants of elg-9 by RT-PCR. The results showed mRNA expression levels that some splice variants of elg-9 are down-regulated after hypoxia. In addition, to further prove the regulation of splice variants of egl-9 in hypoxia response by functional recovery experiments. We construct expression vectors of splice variants of egl-9 and transgenic C. elegans by microinjection. At present, we have been successfully constructed expression vectors and obtained the transgenic C. elegans, and the results lay the foundation for the next experiments.In summary, the study has revealed alternative splicing has an important regulatory role in hypoxia response, laid an important foundation for further study of regulatory mechanism, and provided important clues to study the pathogenesis of disease hypoxia-related.