Single molecule pull down of double strand RNA binding proteins

RNA molecules are transcribed as single stranded naturally, but with most of them forming in to structures composed of duplex regions, loop, bulge or mismatches. RNAs with double stranded regions, or known as double-strand RNAs (dsRNAs). The class of proteins responsible for processing dsRNAs is termed double-stranded RNA binding proteins (dsRBP). In recent decades, an increasing number of reports have shown the role of dsRBP-dsRNA interaction as core strategy in various cellular regulation pathways, including RNA interference, anti-viral immunity, mRNA transport and alternative splicing. However, little is known about the molecular mechanisms underlying the interaction between dsRBPs and dsRNA. Here we examined four human dsRBPs, ADAD2, TRBP, Staufen 1 and ADAR1 `which have various numbers of RNA binding domains expressed in mammalian cells. We applied single molecule pull-down (SiMPull) assay to investigate the intensity of various dsRNA-dsRBP interactions. Our results demonstrate that despite the highly conserved dsRNA binding domains, the dsRBPs exhibit diverse substrate specificy. While TRBP and ADAR1 have a preference for binding simple duplex RNA, ADAD2 and Staufen1 display higher affinity to imperfectly base-paired structured RNA substrates. We also demonstrate ATP-independent sliding activity of TRBP and Staufen probed by single molecule protein induced fluorescence enhancement (smPIFE), which demonstrates how single molecule approaches could be utilized to provide new insight into molecular mechanisms involved in protein-RNA interaction. Collectively, our study highlights the diverse nature of substrate specificity exhibited by dsRBPs that may be critical for their cellular function.

Name thesis:degree_name

M.S.

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Bioengineering

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Wang, Xinlei

Contributors dc:contributor

• Myong, Su-A

• Single molecule
• RNA protein interaction
• double-strand RNA binding protein

Statement dc:rights

• Copyright 2015 Xinlei Wang

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/89193

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/89193