The ability to engineer the specificity of RNA-binding proteins would allow us to modulate gene expression at the post-transcriptional stage, much like re-engineered zinc-finger proteins are used to control transcription. This task has not yet been achieved because methods for the selection of RBPs with altered sequence specificity, such as phage display, are limited in terms of the number of amino acids that can be explored per selection, and computational design methods are still premature. We recently developed a selection method which will allow us to engineer the specificity of RNA-binding proteins based on the in vitro compartmentalization (IVC) approach. These engineered proteins could be used to interrogate gene expression pathways and to perhaps eventually generate new therapeutic proteins as well. I will be using the IVC approach to alter the activity of hnRNP A2. I plan to use this engineered protein to interrogate post-transcriptional gene regulation pathways responsible for mRNA localization in neurons.
mRNA transport in neurons is implicated in synaptic plasticity, memory and several diseases, including fragile X mental retardation syndrome, the most common inherited cause of mental impairment. RNA trafficking is understood to function through the action of RBPs binding to specific RNA sequences, usually located in the 3’ UTR of the mRNA. In particular, the sequence-specific interaction between the heterogeneous nuclear ribonucleoprotein (hnRNP) A2 and the 11-nucleotide A2 response element (A2RE) has been associated with dendritic localization of mRNAs in neurons and oligodendrocytes. Most interestingly, three individual nucleotide substitutions in A2RE have been shown to interfere with binding in vitro and to abrogate mRNA localization in vivo. I intend to engineer a mutant hnRNP A2 protein which recognizes a mutant A2RE in order to specifically interrogate the role of this protein:RNA interaction in vivo.