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. Author manuscript; available in PMC: 2016 Feb 19.
Published in final edited form as: Mol Cell. 2015 Feb 19;57(4):721–734. doi: 10.1016/j.molcel.2015.01.004

Figure 6.

Figure 6

Stress- and p38 MAPK-induced loss of Drosha function. (A) Heat-induced decrease in the levels of endogenous pre-miRNAs. HEK293T cells were treated with SB203580 or vehicle for 30 min, exposed to heat (45°C, 45 min), and re-cultured at 37°C for 3 h. Left panel shows the levels of endogenous pre-miRNAs determined by qRT-PCR in PureLink isolation kit prepared samples. Right panel shows the levels of the corresponding pri-miRNAs determined by qRT-PCR in TRIzol prepared samples (levels of pri- or pre-miRNAs without heat treatment were set as 100%. Reduction: pri-miRs 30b, 26a, and 34a; no change: pri-miRs 16-1, 30a, and 21; increase: pri-miRs 16-2, 143, and 206). Primers used for qRT-PCR are listed in tables in supplementary experimental procedures. (B) Heat-induced loss of endogenous pre- and mature miRNAs. RNA (5 μg) purified by PureLink isolation kit from cells treated as in 6A was analyzed using Highly Sensitive MiRNA Northern Blot Kit (Signosis) and probes (sequences in supplementary experimental procedures). (C) p38 MAPK-induced loss of pri-miR-30a processing in vitro. Internally labeled pri-miR-30a probe was incubated with immunoprecipitated wt Drosha-FLAG (left) or with the total extracts prepared from HEK293T cells transfected as indicated (right). The bottom graphs show the quantification of pre-miR-30a levels (mean ± SEM, n = 3; **p < 0.01 vs Drosha alone group; ##p < 0.01 vs Drosha/p38/MKK6 group). (D) p38 MAPK-dependent loss of pri-miR-30a processing in cells. Left panel: HEK293T cells transfected with pCMV-miR-30a and additional plasmids as indicated were treated with SB203580 for 12 h before the total RNA isolation. The levels of premiR-30a were analyzed by Northern blot with probe for pre-miR-30a (bottom panel shows 5S RNA as loading control). Right panel: Heat-induced loss of pri-miR-30a processing in cells. HEK293 cells transfected with pCMV-miR-30a were treated with SB203580 or vehicle for 30 min and exposed to heat (45°C, 45 min). The levels of pre-miR-30a were determined as in left panel. (E) Resistance to heat-induced inhibition of pri-miR-30a processing by mt5 Drosha. HEK293 cells transfected as indicated were exposed to heat as described in (D). Comparable amounts of Drosha-FLAG were immunoprecipitated and assessed for pri-miR-30a conversion as described in (C). The bottom panel is the quantification of pre-miR-30a levels (mean ± SEM, n = 3; **p < 0.01 vs Drosha alone group). See also Figure S5.