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Biochemistry

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Published: January 16th, 2017

DOI:

10.3791/54391

1Department of Veterinary & Biomedical Sciences, University of Minnesota, 2Department of Veterinary Biosciences, Ohio State University, 3School of Biotechnology, Gautam Buddha University
* These authors contributed equally

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.

The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

Post-transcriptional gene expression is precisely regulated, beginning with DNA transcription in the nucleus. Controlled by RNA binding proteins (RBPs), mRNA biogenesis and metabolism occur in highly dynamic ribonucleoprotein particles (RNPs), which associate and dissociate with a substrate precursor mRNA during the progression of RNA metabolism1-3. Dynamic changes in RNP components affect the post-transcriptional fate of an mRNA and provide quality assurance during the processing of primary transcripts, their nuclear trafficking and localization, their activity as mRNA templates for translation, and the eventual turnover of mature mRNAs.

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Buffer Compositions
Wash Buffer:
50 mM Tris-HCl, pH 7.4
150 mM NaCl
3 mM MgCl2
Low Salt Buffer:
20 mM Tris-HCl, pH 7.5
10 mM NaCl
3 mM MgCl2
2.......

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Prior RIP results identified retroviral gag RNAs and selected cellular RNAs that co-precipitate with DHX9/RHA, including HIV-16, junD6 and huR (Fritz and Boris-Lawrie, unpublished). The retroviral 5' UTR has been demonstrated to co-precipitate with DHX9/RHA in the nucleus and to co-isolate in the cytoplasm on polyribosomes. It is uniquely defined as the cis-acting post-transcriptional control element (PCE)6. To isolate PCEgag RNA-RHA ribonucle.......

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The RNP isolation and cognate RNA identification strategy described here is a selective means of investigating a specific RNA-protein interaction and of discovering candidate proteins co-regulating a specific RNP in cells.

The advantage of using oligonucleotide-directed RNase H cleavage to isolate RNPs is the ability to capture and specifically analyze the RNP cis-acting RNA element over heterogeneous RNPs bound downstream to the cis-acting RNA element of interest. Because the abundance of a c.......

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The authors gratefully acknowledge support by NIH P50GM103297, P30CA100730 and Comprehensive Cancer P01CA16058.

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Name Company Catalog Number Comments
Dynabeads Protein A Invitrogen 10002D
Dynabeads Protein G Invitrogen 10004D
Anti- FLAG antibody Sigma F3165
Anti- FLAG antibody Sigma F7425
Anti-RHA antibody Vaxron PA-001
TRizol LS reagent Life technology 10296-028
RNase H Ambion AM2292
Chloroform Fisher Scientific BP1145-1
Isopropanol Fisher Scientific BP26184
RNaeasy clean-up column Qiagen 74204
Omniscript reverse transcriptase Qiagen 205113
RNase Out Invitrogen 10777-019
Protease inhibitor cocktail Roche 5056489001
Triton X-100 Sigma X100
NP-40 Sigma 98379
Glycerol Fisher Scientific 17904
Random hexamer primers Invitrogen N8080127
Oligo-dT primers Invitrogen AM5730G
PCR primers IDT Gene specific primers for  PCR amplification
Oligonucleotide for RNase H mediated cleavage IDT Anti-sense primer for target RNA
Trypsin Gibco Life technology 25300-054
DMEM tissue culture medium Gibco Life technology 11965-092
Fetal bovine serum Gibco Life technology 10082-147
Tris base Fisher Scientific BP152-5
Sodium chloride Fisher Scientific S642-212
Magnesium chloride Fisher Scientific BP214
DTT Fisher Scientific R0862
Sucrose Fisher Scientific BP220-212
Nitrocellulose membrane Bio-Rad 1620112
Magnetic stand 1.7 ml micro-centrifuge tube holding
Laminar hood For animal tissue culture
CO2 incubator For animal tissue culture
Protein gel apparatus Protein sample separation
Protein transfer apparatus Protein sample transfer
Ready to use protein gels (4-15%) Protein sample separation
Table top centrifuge Pellet down the sample
Table top rotator Mix the sample end to end
Vortex Mix the samples

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