Zachary T. Campbell
University of Texas, USA
Title: Analysis of protein-RNA specificity using a next-generation sequencing approach enables targeted activation of an endogenous human transcript
Biography
Biography: Zachary T. Campbell
Abstract
Targeted control of mRNAs by proteins requires an easily programmable scaffold. PUF domain proteins provide an attractive platform for that purpose. Like TALENs and zinc fingers, which target DNA via reiterated modules, PUF proteins possess simple repeated domains. These target RNA rather than DNA, and so provide an opportunity to control translation, decay and processing of mRNAs. PUF proteins bind to single-stranded RNA using eight repeated modules, each of which contributes three amino acids that contact an RNA base. Here, we identify the specificities of natural and designed combinations of these three amino acids. Our strategy to assay RNA-protein interactions (SEQRS) integrates in vitro selection, high-throughput sequencing of RNA, and SSLs (sequence specificity landscapes) [Campbell ZT et al Cell Reports 2012]. The resulting compendium of specificities reveals the global RNA binding preferences of natural proteins and enables the design of new specificities. Using the recognition code, we design a protein to bind endogenous cyclin B1 mRNA in human cells. A chimeric protein consisting of the designed PUF protein fused to a translation activation domain specifically increases cyclin B1 protein levels, resulting in enhanced sensitivity to chemotherapeutic drugs. Our study provides a guide for rational design of engineered mRNA control, including translational stimulation.