Single molecule pull-down of viral RNA-protein complexes

Protein and RNA molecules interact with multiple protein partners to perform essential cellular processes such as post-transcriptional regulation of mRNA. Recently, a single molecule pull-down (SiMPull) assay was developed to isolate and study single protein complexes directly from cell lysates. Unlike ensemble measurements, SiMPull is a powerful tool that allows detection of diverse proteins present in a single complex and quantitation of the number of interacting partners when the proteins are stoichiometrically labeled. Using a similar principle, the objective of this study is to extend the SiMPull assay to isolate and study single cellular RNA-protein complexes. Utilizing a biological system of virally infected mammalian cells, the substrate targeted in the study are viral RNA-protein complexes. Specifically, the highly replicative sendai virus sub-genomic defective interfering (DI) RNA is targeted. The viral DI RNA associates with multiple viral proteins during replication, and is therefore expected to form heterogeneous RNA-protein complexes. Using the RNA sequence information of DI RNA, we designed several short complementary DNA probes to capture single DI RNA molecules for detection with single molecule fluorescence microscopy. We demonstrate specific capture of viral DI RNA molecules using SiMPull, and could quantitatively measure the presence of interacting viral proteins. Therefore, this study provides evidence for the applicability of SiMPull to isolate and study single cellular RNA-protein interactions.

Name thesis:degree_name

M.S.

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2012

Author dc:creator

• Srinivasan, Divya

Contributors dc:contributor

• Ha, Taekjip

• single molecule
• viral RNA
• total internal reflection fluorescence microscopy
• Ribonucleic acid (RNA)

Statement dc:rights

• Copyright 2012 Divya Srinivasan

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/31924

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/31924