Team:UIUC-Illinois/Project

Abstract

PUF, the magic RNA binding protein: Programmable RNA binding protein with custom functions

         RNA has characteristics that are important in human gene expression (i.e. alternative splicing of mRNA, noncoding RNA). Therefore, a modular RNA binding protein is an invaluable tool for gene regulation. The PUF domain of human PUM1 gene contains eight tandem repeats, each recognizing one of the four nucleotide bases. In theory, a PUF protein can be programmed to recognize any 8-nt ssRNA sequence. Here we demonstrate that PUF can be tethered with other functional domains for applications in E. Coli. Specifically, we show that a PUF/endonuclease fusion protein acts as RNA scissors, silencing gene expression through site specific mRNA cleavage. PUF was also tethered to split GFP to test its ability to co-localize proteins using a RNA scaffold. PUF biobricks offer a wide range of possible functions including gene expression modulation and scaffolding of metabolic pathways.

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PUF Experimental Design

PUF Data

RNA Scaffold Design

RNA Scaffold Data

Biobricks

To read more about our side projects:

Overview

Petrobrick Characterization

Assembly Line

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Subunit Hydrogen Bond Interactions

Fig. 1. Each subunit recognizes one nucleotide. PUF's engineerability is ideal since we already know which three amino acid side chains are required to recognize each of the four nucleotides, A, U, C, or G. In purple is the RNA, and the three amino acid side chains are shown above it. PUF’s potential to bind any conceivable 8 nucleotide sequence is extremely novel. The NYxxQ subunit recognizes U, SYxxR recognizes C, CRxxQ/SRxxQ both recognize A, and SYxxE recognizes G.

Theoretical Applications

Fig. 2 The above figure represents a few of the unlimited possibilities entailed by engineering PUF for applicable functions. Applications involving protein localization to specific RNA sequences is key in engineering the versatility of PUF since varied proteins with varied functions can now be given binding specificity from PUF.