A cohesive strategy is presented to identify small molecule effectors of maternal gene expression. During oogenesis, the cytoplasm of the egg is loaded with silenced maternal transcripts that are activated after fertilization in response to developmental cues. Initial patterning is achieved before nascent transcription begins by activating translation of maternal transcripts at specific times in defined locations. A handful of RNA-binding proteins are responsible for silencing maternal transcripts during oogenesis and coordinating activation after fertilization. Here, we define a strategy to screen for inhibitors of two such proteins, the tandem zinc finger proteins MEX-5 and POS-1. These proteins are critical for anterior and posterior cell fate determination in the nematode worm Caenorhabditis elegans, and are related to tristetraprolin, a factor critical in the regulation of the inflammation response in humans. A process for identifying, counter-screening, and validating small molecule inhibitors is presented that relies on both in vitro fluorescence assays and live imaging during embryogenesis. Inhibitory compounds identified through this screen could serve as """"""""chemical alleles"""""""", enabling the temporal and spatial dissection of maternal gene patterning beyond the earliest points in embryogenesis. The strategy delineated here is generally useful and can be adapted to any RNA-binding protein, opening the door to screening for inhibitors of potentially therapeutic target RNA-binding proteins including tristetraprolin, which may lead to new therapies for rheumatoid arthritis, psoriasis, and other inflammatory diseases including multiple sclerosis. This proposal describes a method for identification and validation of small chemical inhibitors of proteins that bind to RNA. The strategy will be perfected using two protein targets that are critical to differentiation of embryonic stem cells in a worm model. Once optimized, it may be used for therapeutically relevant RNA-binding proteins, including a related protein that is required for deactivating inflammation. This work will have direct relevance to the search for new and better treatments for rheumatoid arthritis, psoriasis, and other inflammatory diseases. Moreover, the lessons learned from this project will contribute to our understanding of the biological processes that allow stem cells to change into all types of adult cells. ?
| Ryder, Sean P; Massi, Francesca (2010) Insights into the structural basis of RNA recognition by STAR domain proteins. Adv Exp Med Biol 693:37-53 |
| Swamidass, S Joshua; Bittker, Joshua A; Bodycombe, Nicole E et al. (2010) An economic framework to prioritize confirmatory tests after a high-throughput screen. J Biomol Screen 15:680-6 |
| Farley, Brian M; Ryder, Sean P (2008) Regulation of maternal mRNAs in early development. Crit Rev Biochem Mol Biol 43:135-62 |
