Project description:MicroRNAs (miRNAs) are 18-24 nucleotide autonomous regulatory RNA molecules found in all eukaryotes. They are involved in the regulation of a multitude of genetic and biological pathways through post transcriptional gene silencing and/or translational repression. Previous data has suggested a slow evolutionary rate for the saltwater crocodile (Crocodylus porosus) over the past several million years when compared to its closest extant relatives, the birds. Understanding genome regulation, adaptive capabilities and physiological features in the saltwater crocodile in the context of relatively slow genomic change thus holds significant potential for the investigation of genomics, evolution and adaptive studies. Utilizing eleven different tissue types and sixteen small RNA libraries, we report a catalog of 644 miRNAs in the saltwater crocodile with > 78% of miRNAs being potentially novel to crocodilians. We also predicted and identified targets for the miRNAs as well as analyzed the relationship of the miRNA repertoire to transposable elements (TEs) in the saltwater crocodile that showed an increased association of DNA transposons with miRNA biogenesis when compared to retrotransposons. Phylogenetic analysis of C. porosus miRNA expectedly revealed highest number of miRNAs in sister crocodilian clades of the American Alligator and the Indian Gharial. This work reports the first comprehensive analysis of miRNAs in Crocodylus porosus for and addresses the potential impacts of miRNAs in regulating the genome in the saltwater crocodile as well as supporting the role of TEs as a source for miRNAs, adding to the increasing evidence that TEs play a significant role in the evolution of gene regulation.
Project description:To study whether increase in mitochondrial oxidative stress (SOD2 removal) and decrease in mitochondrial DNA repair (Ogg1 dMTS) results into increase in mitochondrial DNA mutation load. Oxidative stress has been suggested to induce mutations in mtDNA. To verify this, we extracted and sequenced (Illumina) mitochondrial DNA from heart Sod2 knockout animals that were also deficient for mitochondrial base-excision repair. The repair deficiency was induced by removing the genomic region encoding for the predicted mitochondrial targeting sequence from endogenous OGG1 (L2 to W23) called Ogg1 dMTS mice, thus excluding the protein from mitochondria. OGG1 is a DNA glycosylase that recognizes and repairs 8-oxo-dG damage from DNA. Oxidative stress can induce 8-oxo-dG lesions, thus we removed the mitochondrial matrix localized superoxide dismutase (SOD2) from these mice to increase the level of oxidative stress. 8-oxo-dG lesion can be mutagenic because some DNA repair polymerases are known to erroneously incorporate adenosine opposite to 8-oxo-dG during replication leading to GC>TA transversion mutations.