Using Transcriptome Analysis to Solve the Puzzle of DNA Repair in Bdelloid Rotifers

Abstract

Bdelloid rotifers are microscopic aquatic animals that have apparently survived for more than 40 million years without sex, males, or meiosis. Bdelloids have an efficient DNA repair system that gives them the ability to recover from levels of ionizing radiation that are lethal to other eukaryotes. In bdelloids, DNA double strand breaks induced by high doses (>1000 Gray) of ionizing radiation are repaired without significant loss of viability. The objective of this project is to gain insight into the mechanisms of DNA repair in bdelloids by identifying genes involved in this process. This was done using transcriptome sequencing (RNA‐seq) to characterize genes that are differentially expressed during DNA repair in the bdelloid Adineta vaga. To induce DNA double‐strand breaks, bdelloid cultures were irradiated with a total dose of 280 Gray of ionizing radiation using a cesium‐137 source. RNA was then isolated from these cultures at three time points (0, 30 and 60 minutes) post‐irradiation and also from non‐irradiated controls. RNA‐seq was done using the TruSeq RNA Library Preparation Kit and the Illumina HiSeq 2500 platform. From this data, over 500 genes were differentially expressed during DNA repair. Real‐time PCR was done to validate some differentially expressed genes from RNA‐seq. Gene ontology enrichment analysis was carried out to identify biological processes and pathways that were differentially expressed in irradiated bdelloids. Many upregulated genes were related to DNA repair functions (e.g. ligation, non‐homologous end‐joining, DNA polymerases) while the majority of downregulated genes were associated with transcription, translation and cell cycle regulation. Interestingly, over 20% of differentially expressed genes are apparent “bdelloid‐specific genes,” meaning that they lack homologs in other eukaryotes. However, in some cases these bdelloid‐specific genes represent inadequately annotated gene models; for example, the Random Amplification of cDNA Ends (RACE) method was used to identify a previously unannotated map kinase‐activated protein kinase 2 homolog among these genes. Conversely, other differentially expressed genes appear to be genuine bdelloid‐specific genes since further investigation using computational and molecular methods failed to identify their homolog. Overall, this ongoing gene expression analysis has identified several candidate genes for having functions in DNA repair, which will provide further insight into the mechanism of DNA repair in bdelloid rotifers.Support or Funding InformationThis work was supported by grants from the National Center for Research Resources (5P20RR016460) & National Institute of General Medical Sciences (8P20GM103429) from the NIH.