Team:UIUC-Illinois/Project

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<center><h1>Abstract</h1></center>
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<h2>PUF, the magic RNA binding protein: Programmable RNA binding protein with custom functions</h2>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 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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<object width="600" height="300"><param name="movie" value="http://www.youtube.com/v/TfvAo6tvyLU?version=3&amp;hl=en_US"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/TfvAo6tvyLU?version=3&amp;hl=en_US" type="application/x-shockwave-flash" width="600" height="300" allowscriptaccess="always" allowfullscreen="true"></embed></object></div>
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<p align="left"> Click below to read more about our main project:</p>
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<li><a href="https://2012.igem.org/Team:UIUC-Illinois/Project/Design">PUF Experimental Design</a></li>
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<li><a href="https://2012.igem.org/Team:UIUC-Illinois/Results">PUF Data</a></li>
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                        <li><a href="https://2012.igem.org/Team:UIUC-Illinois/Project/Future/Scaffold">RNA Scaffold Design</a></li>
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<li><a href="https://2012.igem.org/Team:UIUC-Illinois/Results/Scaffold">RNA Scaffold Data</a></li>
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                        <li><a href="https://2012.igem.org/Team:UIUC-Illinois/Results/Biobricks">Biobricks</a></li>
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<br/><br/>
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<p align="left"> To read more about our side projects:
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                <li><a href="https://2012.igem.org/Team:UIUC-Illinois/Project/Future">Overview</a></li>
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                <li><a href="https://2012.igem.org/Team:UIUC-Illinois/Project/Future/Petrobrick">Petrobrick Characterization</a></li>
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                <li><a href="https://2012.igem.org/Team:UIUC-Illinois/Project/Future/AssemblyLine">Assembly Line</a></li>
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<br/><br/>
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<p align="left"> <a href="http://www.youtube.com/channel/UC-PcmkS1Jn9by0h_4uwbDMA">Subscribe</a> to our Youtube channel for upcoming updates with our modeling endeavors!
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<center><h2>Subunit Hydrogen Bond Interactions</h2>
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<img src="https://static.igem.org/mediawiki/2012/3/32/Hbond.png" height=70% width=70%>
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<b>Fig. 1.</b>
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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.
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<center><h2>Theoretical Applications</h2>
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<img src="https://static.igem.org/mediawiki/2012/e/ee/Diagram.png" height=70% width=70%>
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<b>Fig. 2</b>
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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.
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Latest revision as of 00:54, 27 October 2012

Header

Main

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.


Click below to read more about our main project:

  • PUF Experimental Design
  • PUF Data
  • RNA Scaffold Design
  • RNA Scaffold Data
  • Biobricks


    • To read more about our side projects:

  • Overview
  • Petrobrick Characterization
  • Assembly Line


    • Subscribe to our Youtube channel for upcoming updates with our modeling endeavors!

    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.

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