CRISPR guide multiplexing to explore RNA binding Protein Families

Authors

• Q Brail
• A Schmiechan
• P Batista

Abstract

Many diseases are caused by disruptions to regulatory networks, most notably cancer. A central component of gene expression regulation that is still poorly understood is the epitranscriptome, the regulation of RNA function by reversible post-transcriptional modifications. N6-methyladenosine (m6A), the most abundant RNA modification found in mRNA, has been shown to regulate multiple levels of RNA metabolism including RNA splicing, control over translation, and RNA stability. Many cancers have altered levels of m6A, especially aggressive tumors that develop from cancer stem cells, such as gliomas and breast cancers, demonstrating the importance of epitranscriptional regulation in human disease. Here we seek to elucidate how m6A interacting proteins of the YTH family influence mRNA metabolism. These proteins share a YTH domain, which is highly conserved across all eukarya, and allows these proteins to interact with RNA in an m6A dependent manner. Studies on the function of this proteins family in multiple cell types have reached diverse and often contradictory results. To help resolve the function of YTH domain proteins we will employ CRISPR-interference using a multiplex gene editing strategy, generating mutants with different combinations of YTH family proteins loss of function. This will allow us to examine how the m6A modified mRNAs are affected and ultimately determine the degree of redundancy in these m6A interacting proteins. We will further illuminate how this RNA modification contributes to m6A-dependent gene regulation and ultimately, human disease.

Scientific Focus Area: Cancer Biology

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