RNA pulldown assay

RNA pulldown was performed as described previously (46). Briefly, biotin-labeled RNAs were transcribed in vitro using the Biotin RNA Labeling Mix and T7 RNA polymerase (Ambion) and purified with the RNeasy Mini Kit (QIAGEN). YFP coding sequence in a reverse direction (YFP-NC) was used as the control. Folded RNAs (3 μg) were added into 5 mg precleared liver lysates of humanized mice (supplemented with 0.2 mg/mL heparin, 0.2 mg/mL yeast tRNA, and 1 mM DTT) and incubated at 4°C for 1 hour. Then 60 μL of washed streptavidin-coupled Dynabeads (Invitrogen) were added to each binding reaction and further incubated at 4°C for 1 hour. Beads were washed 5 times with lysis buffer (150 mM NaCl, 20 mM Tris pH 7.4, 1 mM EDTA, 0.5% Triton X-100 with Protease/Phosphatase Inhibitor Cocktail and RNaseOUT, Thermo Fisher Scientific) and heated at 70°C for 10 minutes in 1× lithium dodecyl sulfate (LDS) loading buffer, and retrieved proteins were visualized by SDS-PAGE and silver staining. The unique protein bands shown in the hLMR1 RNA pulldown were identified by mass spectrometry analysis at NHLBI Proteomics Facility.

For RNA pulldown using the chromatin lysate, folded RNAs (2 μg) were added into 5 μg liver tissue chromatin lysate of regular mice (supplemented with 0.2 mg/mL yeast tRNA, 0.2 mg/mL DNA from salmon testes) and incubated at 4°C for 1 hour. Then, 30 μL of washed streptavidin-coupled Dynabeads (Invitrogen) were added to each binding reaction and further incubated at 4°C for 1 hour. Beads were washed 5 times with wash buffer (150 mM NaCl, 20 mM Tris pH 7.4, 1 mM EDTA, 0.5% Triton X-100 with Protease/Phosphatase Inhibitor Cocktail and RNaseOUT and 1 mM DTT). The DNA was eluted by digesting the washed beads with RNase A and then treated with proteinase K. The DNA was purified by using the column from Simple ChIP Enzymatic Chromatin IP kit (Cell Signaling Technology). The primers used were the same as used for mouse Ptbp1 ChIP analysis (Supplemental Figure 5).