Abstract
RNA-RNA and RNA-protein interactions are involved in the regulation of gene expression. Here, we describe an updated and extended version of our RNA purification and protein identification (RaPID) protocol for the pulldown of aptamer-tagged mRNAs by affinity purification. The method takes advantage of the high affinity interaction between the MS2 RNA aptamer and the MS2 coat protein (MCP), as well as that between streptavidin-binding peptide (SBP) and streptavidin. Thus, it employs MCP-SBP fusions to affinity purify MS2-tagged target RNAs of interest over immobilized streptavidin. Purified aptamer-tagged mRNAs, along with any associated RNAs and proteins, are then sent for RNA sequencing (RaPID-seq) or mass spectrometry (RaPID-MS), which allows for the identification of bound cohort RNAs and proteins, respectively.
Highlights
[Background] RNA-binding proteins (RBPs) and RNA-associated proteins regulate every step in the gene expression pathway
The advantage of RNA purification and protein identification (RaPID) is the use of the high affinity interaction between the MS2 aptamer and MS2 coat protein (MCP), as well as that of the streptavidin-binding peptide (SBP), which is fused to MCP, to streptavidin beads (Slobodin and Gerst, 2010 and 2011)
Transfer the total amount of protein extract desired for RaPID-seq/MS to a fresh 15 mL tube, add the avidin solution (10 μg of avidin per 1 mg of protein extract), and incubate at 4°C for 1 h with constant rotation/shaking
Summary
2. Transform the strain having the gene tagged with MS2 aptamer with plasmids expressing the MCP-GFP-SBP using standard yeast LiOAc transformation (Gietz and Schiestl, 2007), and plate on SC-His synthetic media agarose plate. Measure the cell number or OD600 of a culture and determine the doubling time using the formula below. [log (Nt/N0)]/log10(2) = g g – number of generations N0 – # of cells or OD600 at start Nt – # of cells or OD600 at the end t = time cultured d – doubling time (d=t/g) b. 6. Resuspend the cell pellet by gentle pipetting (do not vortex) in 200 mL of fresh growth medium lacking methionine, transfer to 1 L flask, and incubate at 30°C for 45 min with shaking to induce the expression of the MS2-CP-GFP-SBP. The cells may be stored for prolonged periods under these conditions
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