Isolation of cognate RNA-protein complexes from cells using oligonucleotide-directed elution

Gatikrushna Singh, Sarah M. Fritz, Arnaz Ranji, Deepali Singh, Kathleen Boris-Lawrie

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

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 stressassociated huR and junD cognate mRNAs.

Original languageEnglish (US)
Article numbere54391
JournalJournal of Visualized Experiments
Volume2017
Issue number119
DOIs
StatePublished - Jan 16 2017

Bibliographical note

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

Publisher Copyright:
© 2017 Journal of Visualized Experiments.

Keywords

  • 5’ untranslated region
  • Biochemistry
  • Cis-acting RNA element and cognate RNA binding proteins
  • DHX9/RNA helicase A
  • Immunoprecipitated RNA binding protein
  • Issue 119
  • Post-transcriptional control
  • RNA-DNA hybrid
  • RNase H cleavage
  • Retrovirus RNA
  • Ribonucleoprotein
  • mRNA targets of RNA binding proteins identified by RNAseq and proteomics

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