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. Author manuscript; available in PMC: 2019 Feb 8.
Published in final edited form as: Cell. 2018 Jan 25;172(4):797–810.e13. doi: 10.1016/j.cell.2017.12.016

Figure 5. Paired Alus, not unpaired Alus, stimulate GOF MDA5 and are abundant in cytosol.

Figure 5

(A–C) Representative electron micrographs (A), IRF3 stimulatory activity (B), and ATPase activity (C) of G495R in complex with the sense (+) or antisense (−) strand of Alu from the NICN1 3’UTR.

(D) Schematic of the RNase H-based method to selectively cleave unpaired, but not paired Alu RNAs.

(E) Gel analysis of the RNase H assay. In vitro transcribed Alu RNAs (from NICN1 3’UTR) were subjected to the RNase H assay as described in (D). An oligo targeting GAPDH (αGAPDH) was used for negative controls.

(F) Quantitation of Alu:Alu hybrids in cytosolic RNA. The RNase H assay in (D) was performed using purified cytosolic RNA from 293T cells, and remaining Alu(+) and Alu(−) were quantitated relative to the spike-in control.

(G) The levels of Alu(+), Alu(−), GAPDH and ACTB (right) relative to the spike-in control before and after the RNase A protection assay. CytoRNA-0.0, −0.5 and −2.0 indicate RNAs recovered after digestion with 0.0, 0.5 and 2.0 ng/µl RNase A in the presence or absence of G495R.

Data represent mean ± SD (n=3) for (C), (F) and (G). See also Figure S5.