Early development in all animals depends initially on the maternal contribution of RNAs and proteins, deposited by the mother into the egg. The genome of the zygote is not actively transcribed initially, and all cellular processes including cel division are mediated by these initial maternal factors. At a later stage, the zygotic genome is activated and control switches from maternally contributed factors to newly synthesized zygotic RNAs and proteins. A key step for subsequent development is the destruction of maternal components. While this process has been known for over three decades, the molecular elements required to clear maternal instructions during vertebrate development remain largely unknown. In this proposal, I will investigate the mechanisms of maternal RNA degradation in vertebrates, using zebrafish as a model system.
In Aim 1, I will use high throughput sequencing to identify maternally derived transcripts with correlated degradation patterns. I will compare timecourse expression data in wildtype embryos as well as embryos in which de novo transcription and small regulatory RNA synthesis have been inhibited, in order to define transcripts undergoing degradation by specific mechanisms and under specific temporal control.
In Aim 2, I will analyze the lengths and sequence content of the 3'untranslated regions (UTRs) of maternal transcripts and look for regulated differences between the 3'UTRs of zygotic transcripts, under the hypothesis that differential regulation and degradation is mediated by these sequences.
In Aim 3, I will characterize the discrete regulatory sequence signals present in 3'UTRs that are the determinants of degradation, using a combination of computational methods and reporter gene constructs. I expect that maternal RNAs that are co-degraded should also contain the same sets of signals. Together these aims will elucidate the ways in which RNA transcripts are differentially degraded. Although these processes are especially prominent during development, they are relevant to all living cells, which need to maintain regulated quantities of RNA messages in order to achieve specific phenotypes. Thus, understanding the mechanisms underlying RNA degradation is key to understanding how this process is misregulated in developmental disorders, cancers, and disease.
Early development in animals depends on the strict temporal control of gene messages, specifically which messages are allowed to be used to generate proteins, and which are no longer necessary and marked for destruction. Although this phenomenon is a hallmark of embryogenesis, during which time the protein requirements of the embryo rapidly change, it is in fact ubiquitous to all living cells. Thus, understanding the mechanisms by which messages are destroyed, as well as the signals that mark them for destruction, is fundamental to understanding and developing treatments for diseases caused by the improper regulation of gene message quantities, such as cancer.
|Bazzini, Ariel A; Johnstone, Timothy G; Christiano, Romain et al. (2014) Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation. EMBO J 33:981-93|
|Lee, Miler T; Bonneau, Ashley R; Giraldez, Antonio J (2014) Zygotic genome activation during the maternal-to-zygotic transition. Annu Rev Cell Dev Biol 30:581-613|
|Lee, Miler T; Bonneau, Ashley R; Takacs, Carter M et al. (2013) Nanog, Pou5f1 and SoxB1 activate zygotic gene expression during the maternal-to-zygotic transition. Nature 503:360-4|