Single molecule pull-down of viral RNA-protein complexes

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

Srinivasan, Divya ; Ha ; Taekjip

关键词: single molecule;    viral RNA;    total internal reflection fluorescence microscopy;    Ribonucleic acid (RNA);   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/31924/Srinivasan_Divya.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Protein and RNA molecules interact with multiple protein partners to performessential cellular processes such as post-transcriptional regulation of mRNA. Recently,a single molecule pull-down (SiMPull) assay was developed to isolate and study singleprotein complexes directly from cell lysates. Unlike ensemble measurements, SiMPull isa powerful tool that allows detection of diverse proteins present in a single complex andquantitation of the number of interacting partners when the proteins arestoichiometrically labeled. Using a similar principle, the objective of this study is toextend the SiMPull assay to isolate and study single cellular RNA-protein complexes.Utilizing a biological system of virally infected mammalian cells, the substrate targetedin the study are viral RNA-protein complexes. Specifically, the highly replicative sendaivirus sub-genomic defective interfering (DI) RNA is targeted. The viral DI RNAassociates with multiple viral proteins during replication, and is therefore expected toform heterogeneous RNA-protein complexes. Using the RNA sequence information ofDI RNA, we designed several short complementary DNA probes to capture single DIRNA molecules for detection with single molecule fluorescence microscopy. Wedemonstrate specific capture of viral DI RNA molecules using SiMPull, and couldquantitatively measure the presence of interacting viral proteins. Therefore, this studyprovides evidence for the applicability of SiMPull to isolate and study single cellularRNA-protein interactions.