The function of miR-31 and its targets during early development
Date
2023
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
miR-31 is a highly conserved microRNA that has been well studied in the context of cancer, but its role in development is not well-established. Using the sea urchin as a model, we identified that miR-31 regulates early development. miR-31 inhibited embryos exhibit defects in chromosome segregation, increased microtubules and increased F-actin compared to control embryos in early cleavage stages. In order to understand the mechanism of these defects, we identified several of miR-31’s direct targets. We validated miR-31’s regulation of Fascin, an actin-bundling protein, Rab35, a small GTPase that mediates actin polymerization, Gelsolin, an actin capping and severing protein, as well as β-actin, an actin isoform. ☐ To understand how miR-31’s regulation of Rab35 affects development, we identified the function of Rab35 during development. We found that Rab35 plays an important role in gastrulation and skeletogenesis through its ability to mediate both actin dynamics and endosomal trafficking. Additionally, we found that Fascin overexpression rescues Rab35 KD phenotypes, indicating that Rab35’s functional interaction with Fascin is evolutionarily conserved. ☐ In addition, we identified that Fascin, another target of miR-31, regulates gastrulation and skeletogenesis. We propose several experiments to understand the mechanism of Fascin’s regulation of these important processes. We show that Fascin is important for early cleavage stage development, and that the localization of its translation plays an important role during this crucial stage of development. ☐ Along with the observation that miR-31 and its target transcripts are localized to the mitotic spindle, we identified other important mitotic transcripts to be localized there as well. This localization of several of these transcripts to the mitotic spindle is conserved in mammalian cells as well. Disruption of actin dynamics does not alter the localization of these transcripts, but the localization of these transcripts is dependent upon microtubule dynamics. Additionally, both kinesin-1 and dynein play roles in the localization of the transcripts of these mitotic genes. The 3’UTR of one of these genes, Aurora B, contains a CPE which is essential for its localization to the mitotic spindle. Disrupting the ability of CPEB to bind to this region of the RNA results not only in lack of localization of Aurora B to the mitotic spindle, but also results in developmental delay and arrest in early cleavage stage embryos. This suggests that localization of the Aurora B transcript to the mitotic spindle plays an important role in development. ☐ To continue investigating the role that transcript localization plays, we observed that both miR-31 and its target transcripts localize to the mitotic spindle in early cleavage stage embryos, pig kidney cells and human colon cancer cells, indicating that their localization to the spindles is evolutionarily conserved. We also observed newly translated protein and translational elongation factors at the mitotic spindle. Using a new technique, Puro-PLA, we observed newly translated Fascin protein at the mitotic spindle in both human cells and sea urchin embryos, suggesting that Fascin is being translated at the mitotic spindle. Consistent with this, miR-31 inhibition results in more Fascin and Rab35 protein, both direct targets of miR-31, at the mitotic spindle. Forcing Fascin RNA to be translated at the cell cortex results in developmental delay and arrest in early cleavage stage embryos, suggesting that local translation of Fascin at the mitotic spindle is essential for proper development. As miR-31 is an evolutionarily conserved miRNA, and we have shown that the localization of miR-31 and its targets is also conserved, identifying the function of miR-31 in early development using the sea urchin reveals a deeper understanding of the regulatory processes that occur during development in animal embryos.
Description
Keywords
Fascin, MicroRNA, Mitosis, RNA localization, Sea urchin