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.
Bibliographical noteFunding Information:
The authors gratefully acknowledge support by NIH P50GM103297, P30CA100730 and Comprehensive Cancer P01CA16058.
© 2017 Journal of Visualized Experiments.
- 5’ untranslated region
- 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
- mRNA targets of RNA binding proteins identified by RNAseq and proteomics