The mir-35 family of microRNAs are developmentally regulated in Caenorhabditis elegans and are maternally contributed as well as zygotically expressed in early embryos. Expression of the mir-35 family is essential for viability; complete loss of the mir-35 family results in lethality. The mir-35 family is tightly regulated at various stages during C. elegans development; mir-35 family is haploinsufficient for the regulation of sex determination indicating that the embryonic steady-state abundance is tightly controlled. In addition, mir-35 family abundance is developmentally regulated and is sharply decayed at the end of embryogenesis. Because of the tight regulation of the mir-35 family at steady-state and during development, this family of miRNAs is a strong candidate for studying the mechanisms of miRNA decay. The mir-35 family has two defining characteristics: the first is a shared, family-specific seed sequence, and second, mir-35 is preferentially loaded into the Argonaute protein, ALG-2. We are interested in exploring both of these characteristics to ascertain their roles in the stability or decay of the mir-35 family. To examine if the mir-35 family turnover is seed sequence-specific, we mutated the seed sequence of the mir-35 miRNA and monitored mir-35 levels past embryogenesis. We detected a perdurance of mir-35 levels past embryogenesis indicating that mir-35 family turnover is seed sequence-specific. Reciprocally, we are interrogating whether the 3' end of mir-35 plays a critical role in the stability or decay of the mir-35 family by mutating mir-35 outside of the seed region and examining the temporal expression of mir-35. Additionally, we seek to explore the role of ALG-2 in the stability of the mir-35 family. Strains containing tagged ALG-1 or ALG-2 in a background with the mir-35 seed mutants will be analyzed by immunoprecipitation followed by qPCR. These experiments will determine whether the delayed decay of mir-35 seed mutants is due to aberrant loading of the microRNA (e.g. loading into ALG-1). Overall, we aim to understand the regulation of mir-35 family abundance in early development and the role of the mir-35 family in C. elegans. By elucidating the mechanisms of the mir-35 family decay, this research will offer insight into the broader mechanisms regulating miRNA steady state abundance and turnover.

Affiliations:
- Department of Biology, Johns Hopkins University, Baltimore, MD, USA
- Laboratory of Cellular and Developmental Biology, NIDDK, NIH, Bethesda, MD, USA