Bhat, Jaffar M. et al. (2019) International Worm Meeting "Unraveling the function of circRNAs in C. elegans aging."

Viramontes, David, Bhat, Jaffar M., Grubbs, Jeremy J., Knupp, David, Miura, Pedro, Nikooei, Tatiana, van der Linden , Alexander M.

[International Worm Meeting, 2019]

Circular RNAs (circRNAs) are an enigmatic species of endogenous RNAs. They are highly stable RNA molecules mostly generated by back-splicing events from known protein-coding genes, and are abundantly expressed in many animals, from C. elegans to humans. Similar as in mice and fruit flies, circRNAs accumulate during aging on a genome-wide level in C. elegans (BMC Genomics, Issue 19:8, 2018). These trends suggest age-accumulation of circRNAs might influence lifespan. CircRNAs that are highly accumulated include those derived from genes that play a conserved role in lifespan regulation in both mammals and in C. elegans. No function is known for these age-accumulated circRNAs, or for most of the thousands of circRNAs found throughout the animal kingdom. We therefore decided to devise a strategy to delete individual circRNAs in C. elegans in order to study their function in organismal aging. Due to the fact that most circRNAs are encoded from the same exons that produce linear RNAs, a major challenge to study circRNA function is to reduce or eliminate individual circRNAs without affecting expression of linear RNAs from the host gene. Moreover, due to the mechanism of RNAi in C. elegans, we cannot use siRNAs to specifically knockdown circRNAs and not their linear counterparts. To circumvent these issues, we identified Reverse Complementary base pair Matches (RCMs) in intron sequences flanking circRNAs that are predicted to promote circularization. Targeting an RCM of the crh-1 circRNA using CRISPR/Cas9 led to successful deletion of the crh-1 circRNA without disrupting the mRNA expression from the host crh-1 gene, which encodes for CREB. Transgenic overexpression of the crh-1 circRNA under control of the pie-1 promoter strongly increased crh-1 circRNA expression, and was able to restore the loss of crh-1 circRNA expression in the crh-1 circRNA mutant. To our knowledge, these findings represent the first genetic mutant of a circRNA in any animal. Preliminary analysis shows that the mean life-span of crh-1 circRNA mutant animals is longer than wild-type, suggesting that the crh-1 circRNA plays a role in the aging process.

Jolanta Polanowska et al. (2006) European Worm Meeting "Regulation of BRC-1 - Dependent Ubiquitylation at DNA Damage Sites"

Jolanta Polanowska, Simon Boulton, Tatiana Garcia-Muse, Julie Martin

[European Worm Meeting, 2006]

Jolanta Polanowska1,2, Julie Martin1, Tatiana Garcia-Muse1 and Simon Boulton1. The BRCA1 tumour suppressor and its heterodimeric partner BARD1 constitute an E3-ubiquitin (Ub) ligase and function in DNA repair by unknown mechanisms. We have previously described C. elegans BRCA1 and BARD1 orthologues (brc-1 and brd-1, respectively) that possess many of the functional domains present in their human counterparts, including RING, ankyrin, and BRCT domains (Boulton et al., 2004). Consistent with conserved roles in DNA repair, BRC-1 and BRD-1 interact to form a heterodimer via their respective RING domains. To explore the mechanistic role of BRC-1 and BRD-1 in DNA repair processes we have characterized a C. elegans BRC-1/BRD-1 complex (CeBCD) purified by tandem immunoaffinity before and at different time points after IR-treatment. This approach is a first for C. elegans and demonstrates that protein complexes purified in this manner are amenable to biochemical analysis and can be used in combination with genetics and cell biology to accelerate functional discoveries. We present evidence that the CeBCD complex possesses an E3-Ub ligase that is activated on chromatin in response to IR-treatment and further demonstrate that the DNA damage checkpoint promotes association of the CeBCD complex with E2-Ub conjugating enzyme, Ubc5(LET-70), to form an active E3-Ub ligase in response to DNA damage. We also show that ubiquitylation events at DNA damage sites require brc-1, brd-1, ubc5(let-70), mre-11 and atl-1, thus providing in vivo evidence to support our biochemical analysis.. Boulton, S.J., Martin, J.S., Polanowska, J., Hill, D.E., Gartner, A. and Vidal, M. (2004) Curr Biol, 14, 33-39.