Figure 2.

The Integrator complex is present at the MtnA locus during copper stress and attenuates MtnA transcription. (A) DL1 cells were treated with dsRNAs for 3 d to induce RNAi and depletion of the indicated factors. A total of 500 µM CuSO₄ was added for the last 14 h. RT-qPCR was then used to measure the pre-mRNA levels of endogenous RpL32, DCP2, and MtnA. Primer pairs that span an intron–exon boundary were used to specifically amplify pre-mRNAs. Data were normalized to RpL32 mRNA expression and are shown as mean ± SD, N = 3. (*) P < 0.05. (B) DL1 cells were unstressed (–) or treated with 500 µM CuSO₄ or 50 µM CdCl₂ for 14 h, and RT-qPCR was then used to measure MtnA pre-mRNA levels. Data were normalized to RpL32 mRNA expression and are shown as mean ± SD, N = 3. (*) P < 0.05. (C) DL1 cells were treated with dsRNAs for 3 d and 500 µM CuSO₄ or 50 µM CdCl₂ was added for the final 14 h, as indicated. RT-qPCR was then used to measure MtnA pre-mRNA levels. Data were normalized to RpL32 mRNA expression and are shown as mean ± SD, N = 3. (*) P < 0.05. (D) The MtnA locus with the locations of ChIP amplicons. Recruitment of IntS1 and IntS12 in unstressed cells (gray) or after the cells had been treated with CuSO₄ (blue) or CdCl₂ (green) for 14 h was measured using ChIP-qPCR. Data are shown as fold change relative to the IgG control (mean ± SD, N = 3). (*) P < 0.05.