Figure 6.

RNAP III is responsible for coligo transcription, which appears to take place mainly in the cytosol of transfected cells. (A) α-Amanitin inhibits coligo transcription at concentrations consistent with RNAP III transcription. HEK293T WCE IVT was carried out with increasing concentrations of α-amanitin. C, coligo 19aTAR, 100 nM. Lanes 3–5: 0.12, 1.2, 120 µg/ml α-amanitin. Rel. SRT: relative amount of single round transcript (aka monomer transcript). (B) Northern blot of total RNA from HEK293T cells transfected with 40 nM coligo 19aTAR with concurrent α-amanitin treatment (Lanes 3–5: 0.12, 1.2, 40 µg/ml). 7SK RNA was probed as a loading control. (C) Northern blot of total RNA from HEK293T cells transfected with 40 nM coligo 19aTAR with concurrent RNAP III-specific inhibitor ML-60218 treatment at 68 µM. DMSO, inhibitor solvent. U2 snRNA was probed as loading control. (D) DPAGE of ³²P tracer-labeled coligo 19aTAR recovered from HEK293T transfection after separation of nuclear and cytosolic fractions. Equal percentages of the nuclear and cytosolic fractions were loaded. Inset: western blot assessment of fractionation, β-tub., β-tubulin (cytosolic protein). H4, histone H4 (nuclear protein). M, markers and input templates. (E) Northern blot of RNA isolated from HEK293T nuclear and cytosolic fractions 24 h after transfection with 40 nM coligo 19aTAR. Equal percentages of the nuclear and cytosolic fractions were loaded. Inset: western blot assessment of fractionation, as in panel D. (F) HEK293T nuclear and cytosolic extract IVT in the presence of increasing α-amanitin (α, lanes 3–5 and 10–12: 0.12, 1.2 and 120 µg/ml), DMSO (D) or ML-60218/DMSO solution (ML). Inset: western blot assessment of fractionation, β-tub., β-tubulin (cytosolic protein). CstF-77, (nuclear protein); RPC2, RNAP III subunit.