Lerner, N. | bioRxiv Science

bioRxiv2020

Oocyte Aging is Controlled by Mitogen Activated Protein Kinase Signaling

Oogenesis is one of the first processes to fail during aging. In women, most oocytes cannot successfully complete meiotic divisions during the fourth decade of life. Studies of the nematode Caenorhabditis elegans have uncovered conserved genetic pathways that control lifespan, but our knowledge regarding reproductive aging in worms and humans is limited. Specifically, little is known about germline internal signals that dictate the oogonial biological clock. Here, we report a thorough characterization of the changes in the worm germline during aging. We found that shortly after ovulation halts, germline proliferation declines, while apoptosis continues, leading to a gradual reduction in germ-cell numbers. In late aging stages, we observed that meiotic progression is disturbed and crossover designation and DNA double-strand break repair decrease. In addition, we detected a decline in the quality of mature oocytes during aging, as reflected by decreasing size and elongation of interhomolog distance, a phenotype also observed in human oocytes. Many of these altered processes were previously attributed to MAPK signaling variations in young worms. In support of this, we observed changes in activation dynamics of MPK-1 during aging. We therefore tested the hypothesis that MAPK controls oocyte quality in aged worms using both genetic and pharmacological tools. We found that in mutants with high levels of activated MPK-1, oocyte quality deteriorates more rapidly than in wild-type worms, whereas reduction of MPK-1 levels enhances quality. Thus, our data indicate that MAPK signaling controls germline aging and could be used to attenuate the rate of oogenesis quality decline.

cell biology↗

bioRxiv2018

An in vivo knockdown strategy reveals multiple functions for circMbl

Circular RNAs (circRNAs) are highly abundant and evolutionary conserved RNAs of mostly unknown functions. circRNAs are enriched in the brain and accumulate with age in flies, worms and mice. Despite their abundance, little is known about their functions, especially in the context of whole organisms. Here we report the development and use of shRNAs to knock down and study the function of circMbl, the most abundant circRNA in Drosophila. This circRNA is highly conserved through evolution and is generated from the locus of the essential splicing factor muscleblind (mbl). Briefly, we generated flies in which circMbl is reduced more than 90% without measurable off-target effects in the hosting gene as well as in other RNAs. These flies display specific defects that suggest roles of circMbl in muscle and neural tissues during development and in adult flies. More specifically, whole organism downregulation of circMbl leads to male developmental lethality, altered gene expression, behavioral defects, wing posture- and flight defects. Moreover, these phenotypes are recapitulated by a second shRNA targeting circMbl. Importantly, knockdown and overexpression of circMbl affect mostly the same genes but in the opposite direction. Last but not least, downregulation of circMbl in the fly central nervous system caused abnormal synaptic function. Together, our results demonstrate the functionality of circMbl at the organismal level likely by acting in multiple tissues. Moreover, here we provide the first proof of functionality of circRNAs in Drosophila as well as a methodological approach that enables the comprehensive study of circRNAs in vivo.\n\nSIGNIFICANCE STATEMENTCircular RNAs (circRNAs) are highly abundant and evolutionary conserved RNAs of mostly unknown functions. Here we report the development and use of a shRNA-based system to knockdown specific circRNAs in vivo. We generated flies in which circMbl, the most abundant circRNA is reduced more than 90% without measurable off-target effects. These flies display male developmental lethality, altered gene expression, behavioral defects, wing posture- and flight defects. These phenotypes are recapitulated by a second shRNA targeting circMbl. Moreover, downregulation of circMbl in the fly central nervous system caused abnormal synaptic function. Together, our results demonstrate the functionality of circMbl at the organismal level and provide a methodological approach that enables the comprehensive study of circRNAs in vivo.

molecular biology↗