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. 2017 Mar 20;7(6):e2193.
doi: 10.21769/BioProtoc.2193.

RNA-protein UV-crosslinking Assay

Dipak Kumar Poria  1 , Partho Sarothi Ray  1
Affiliations

RNA-protein UV-crosslinking Assay

Dipak Kumar Poria et al. Bio Protoc. .

Abstract

RNA-protein interactions play a crucial role in every aspect of RNA metabolism, and also plays a major role in post-transcriptional gene regulation. RNA-binding proteins have been implicated in viral gene expression (Ray and Das, 2002) and microRNA-mediated gene regulation (Poria et al., 2016). Here we have described the protocol which (1) covalently links transiently interacting RNA-protein complexes by UV crosslinking, (2) removes the unprotected RNA by RNase digestion and (3) detects the RNA-protein complexes by SDS-PAGE analysis. This protocol provides a rapid and reliable means to directly assay RNA-protein interactions and their kinetics using purified proteins and also help in identifying novel RNA-protein interactions.

Keywords: RNA-binding proteins; RNA-protein interaction; UV-crosslinking.

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Figures

Figure 1.
Figure 1.. Preparation of template DNA for transcription using T7 promoter
Figure 2.
Figure 2.. Preparation of DNA template for oligo-driven transcription
Figure 3.
Figure 3.. UV-crosslinking of 32P labelled PDCD4-3’UTR RNA with 500 ng of purified 6x His-tagged HuR protein (lane 1) and in the presence of 10x (lane 2) and 100x unlabelled PDCD4 3’UTR RNA.
RNA-protein complexes were digested with RNase A and resolved in 10% SDS-PAGE and exposed for phosphorimaging. The HuR-32P PDCD4-3’UTR RNA complexes are indicated by the arrow.
See this image and copyright information in PMC

References

    1. Poria D. K., Guha A., Nandi I. and Ray P. S.(2016). RNA-binding protein HuR sequesters microRNA-21 to prevent translation repression of proinflammatory tumor suppressor gene programmed cell death 4. Oncogene 35(13): 1703-1715. - PMC - PubMed
    1. Ray P. S. and Das S.(2002). La autoantigen is required for the internal ribosome entry site- mediated translation of Coxsackievirus B3 RNA. Nucleic Acids Res 30(20): 4500-4508. - PMC - PubMed