Functional characterisation of the RIO kinase family

The riok family of atypical protein kinases are evolutionary conserved; however their precise functions remain unknown. riok-1 and riok-2 exist in all eukaryotes and have predicted roles in cell cycle regulation and rRNA biogenesis, while riok-3 exists exclusively in metazoans with little known about its function. The riok family were examined in the C. elegans model system. Expression of riok-1, -2, -3 was highest in the first larval stage, consistent with the riok-1 and riok-2 deletion mutants early larval arrest phenotype. To investigate the tissue and developmental expression of RIOK in vivo, GFP tagged transgenic animals were created. RIOK-1 was expressed in a range of different tissues, including the spermatheca, intestinal cells and a subset of neurons. RIOK-2 displayed expression in the pharynx, while riok-3 animals revealed expression in a small subset of neuron. RNAi was used to further analysis riok members, as it bypassed the genetic mutant arrest stage. Our data identified a new novel role for riok-l in oogenesis. Interestingly, knockdown of riok-1 in hermaphrodite animals results in 100% sterility, while males produce viable/functional sperm. Adult riok-1 (RNAi) hermaphrodite gonads are smaller in size and display endomitotic oocytes, where large DNA aggregates were observed. Interestingly, riok-1 knockdown resulted in increased MAP kinase activation in gonads, which has previously been shown to induce endomitotic oocyte formation. To assess the somatic vs. gonad contribution towards the riok-1 (RNAi) defect, riok-1 was knocked down in a genetic background where gonad RNAi only occurs. A major impact on the reproductive capacity of these animals was still apparent, suggesting riok-1 is primarily a germ cell mediated defect. Therefore, riok-1 and riok-2 are required for normal development, with riok-1 possessing a major function in oogenesis. Our data suggests that riok-1 plays a role in meiosis and has an important cell cycle regulator role; however a mechanistic understanding remains to be elucidated.