Native RNA G quadruplex immunoprecipitation (rG4IP) from mammalian cells

Implications of differential transcription start site selection on chronic myeloid leukemia and prostate cancer cell protein expression

“…Therefore, we developed the rG4IP technique to selectively enrich cytosolic RNAs with G4s, not fixing the cells and incorporating a step to degrade any trace amount of genomic DNA. 31 Using circular dichroism and rG4IP, we were able to demonstrate the formation of a G4 structure in AGAP2 longer 5′ UTR ( Figure 3 ). And, as described for other mRNAs, 41 , 55 this structure was responsible for a reduced protein expression ( Figures 4 B and 4C) and a reduced polysome association ( Figure 4 D).…”

“… (B) Overview of the RNA G quadruplex immunoprecipitation (rG4IP) technique. 31 Cells were treated with 25 μg/mL digitonin and the extracted cytoplasmic fraction precleared and incubated overnight with a structure-specific G4 antibody (BG4) bound to protein G magnetic beads. After incubation, the beads were washed, and the bound RNA eluted by unfolding the G4 by heating it at 65°C for 15 min.…”

“… 40 Using this antibody, we developed an RNA-specific G4 immunoprecipitation technique (rG4IP) to selectively enhance the detection of cytosolic mRNAs with G4 structures ( Figure 3 B). 31 …”

“…We demonstrated the formation of the G4 using circular dichroism and an in-house developed immunoprecipitation approach that we have termed rG4IP ( R NA G4 I mmuno p recipitation). 31 We also determined that the presence of the G4 in AGAP2 5′ UTR has a direct impact on the translation efficiency, reducing the amount of mRNA associated with polysomes. But more importantly, we hypothesized that this differential TSS selection could be a more widely used mechanism to regulate the amount of protein produced in cells.…”

“…We used 5' RACE to determine the transcription start sites in prostate cancer and CML cell lines, finding that the transcripts with a slightly longer 5' UTR contained the consensus sequence for a G quadruplex (G4), a type of secondary structure. We demonstrated the formation of the G4 using circular dichroism and an in-house developed immunoprecipitation approach that we have termed rG4IP (RNA G4 Immunoprecipitation) 31 . We also determined that the presence of the G4 in AGAP2 5' UTR has a direct impact on the translation efficiency, reducing the amount of mRNA associated to polysomes.…”