Programmable depletion and rescue platform to screen dynamic regulatory events during cellular differentiation.

PROJECT SUMMARY: The mechanisms by which stem cells orchestrate their program to become functional differentiated cells require accurate temporal regulation of specific gene expression programs. This complex network requires precise temporal regulation of transcription and degradation processes to activate specific programs in a coordinated manner. So far, most of the studies have explored the regulation of transcriptional pathways and chromatin remodeling events during the differentiation process. mRNA degradation processes may present an attractive and still poorly explored opportunity for enhancing our understanding of the differentiation process. However, the lack of technologies that can capture rapid mRNA degradation events over highly dynamic processes, such as differentiation, and the heterogeneity of the mRNA degradation machinery in composition and expression patterns during differentiation have presented major technical limitations to further exploring the role of mRNA degradation across the continuum of the differentiation program. Here I propose to explore the existence of specialized RNA degradation complexes that control the decay of specific mRNA subclasses at precise timeframes of the differentiation process. To test this, we will introduce a new platform that uses cutting-edge technologies integrated in an innovative way to interrogate the continuum of the differentiation process at an unprecedented resolution. Our programmable depletion and rescue strategy will allow us to control the expression level of each subunit of complex mRNA degradation machinery robustly and with a precise time resolution of hours. By combining this technology with a high-content imaging system, we can record phenotype changes and accurately determine the specific impact of any perturbed protein on differentiation. Additionally, the use of this platform will guide us to understand the exact gene regulatory network controlled by the machinery at the transcriptional and stability level. The conceptualization and development of this workflow have the potential to impact a broader scientific audience; due to its extremely high flexibility, it could be applied to the study of unlimited biological processes or proteins. In this essay, the application of our proposed platform has the potential to fundamentally overturn the current view of how mRNA decay is dynamically regulated, providing a definite understanding of the function of the degradation machinery on mRNAs and, at the same time, revealing the broader impact of the degradation process on differentiation.