RNA Polymerase II Activity of Type 3 Pol III Promoters

Abstract

In eukaryotes, three RNA polymerases (Pol I, II, and III) are responsible for the transcription of distinct subsets of genes. Gene-external type 3 Pol III promoters use defined transcription start and termination sites, and they are, therefore, widely used for small RNA expression, including short hairpin RNAs in RNAi applications and guide RNAs in CRISPR-Cas systems. We report that all three commonly used human Pol III promoters (7SK, U6, and H1) mediate luciferase reporter gene expression, which indicates Pol II activity, but to a different extent (H1 ≫ U6 > 7SK). We demonstrate that these promoters can recruit Pol II for transcribing extended messenger transcripts. Intriguingly, selective inhibition of Pol II stimulates the Pol III activity and vice versa, suggesting that two polymerase complexes compete for promoter usage. Pol II initiates transcription at the regular Pol III start site on the 7SK and U6 promoters, but Pol II transcription on the most active H1 promoter starts 8 nt upstream of the Pol III start site. This study provides functional evidence for the close relationship of Pol II and Pol III transcription. These mechanistic insights are important for optimal use of Pol III promoters, and they offer additional flexibility for biotechnology applications of these genetic elements.

Keywords: Pol II; Pol III; dual-polymerase activity; promoter competition; type 3 Pol III promoters.

Figure 1

Small RNA and Luc Reporter Expression by Three Human Type 3 Pol III Promoters (7SK, U6, and H1) (A) Upper panel: P-N44 constructs encode Pol III promoters for transcription of an artificial 44-nt sequence, which terminates at the T6 signal. Middle panel: equimolar amounts of P-Luc constructs were transfected into HEK293T cells and total cellular RNA was harvested 36 hr post-transfection. The pBluescript (pBS) plasmid was used as a negative control. An equal amount of total RNA was subjected to northern blot for detection of the N44 RNA. M, RNA size marker (nt) is shown on the left. Ethidium bromide staining of rRNAs (5S and 5.8S) and tRNA serve as loading controls. Lower panel: quantitation of N44 RNA in middle panel. The N44 transcripts from the respective promoters were normalized to 5S RNA. 7SK-produced N44 was arbitrarily set at 1.0. The northern blot in (B) was repeated twice and very similar results were obtained. (B) Upper panel: design of the P-Luc constructs. Four promoters were inserted in the pGL3-basic backbone to drive Luc gene expression. The P(−) construct without any promoter was used as a negative control. Middle panel: Luc reporter activity of the respective promoters. Equimolar amounts of the P-Luc constructs and 1 ng Renilla plasmid to control for transfection efficiency were co-transfected into HEK293T cells. Dual-luciferase reporter assays were performed 36 hr after transfection, and the ratio of Firefly and Renilla luciferase was calculated to represent the Luc activity. The Luc activity measured for the SV40 promoter was arbitrarily set at 10. The results are presented as mean ± SD (n = 3). Lower panel: quantification of Luc mRNA made by different promoters. Total cellular RNA from P-Luc-transfected HEK293T cells was subjected to qPCR to quantitate the Luc mRNA level. The Luc RNA level for SV40 was arbitrarily set at 10. The GAPDH signal was used as an internal control. The data are shown as the mean ± SD (n = 3). (C) Luc expression in HCT116 and C33A cells and PBMCs. Luciferase assays for HCT116 and C33A cells were performed as in Figure 1B. The Luc activity measured for the SV40 promoter was arbitrarily set at 10. Equimolar amounts of P-Luc constructs were nucleotransfected into an equal number of PBMCs. After 24 hr, the firefly luciferase was measured and relative luminescence values were plotted.

Figure 2

Pol III-Mediated Luc Expression: Sensitivity to Pol II/III Termination Signals (A) Theoretical full-length Luc mRNA level expressed by Pol II and III. Pol III termination signals (T_n stretch with n ≥ 4) in the Luc-coding sequence are indicated. Luc expression was predicted based on the Pol III termination efficiency by these T-stretch signals. (B) Luc constructs with added termination signals (T6 for Pol III and pA for Pol II). Signals were inserted between the promoter and Luc gene to create P-T6-Luc and P-T6-pA-Luc. (C) All three sets of constructs for 7SK, U6, and H1 promoters were transfected into HEK293T cells, and the Luc activity was determined as in Figure 1E.

Figure 3

Luc Expression from Both Pol II and Pol III Promoters Is Sensitive to α-Amanitin (A) The influence of α-amanitin on SV40-mediated Luc expression. The P(SV40)-Luc construct was transfected into HEK293T cells, and cells were treated with α-amanitin for 0, 12, 24, or 36 hr at different concentrations (1, 5, or 10 μg/mL). The Luc activity was measured 36 hr after transfection. Luc activity without α-amanitin (0 hr of treatment) was arbitrarily set at 100%. (B) α-Amanitin significantly inhibits Luc activity and Luc RNA expression of Pol II/III promoters. The four P-Luc constructs were individually transfected with the pRL plasmid into HEK293T cells with or without 2 μg/mL α-amanitin. The dual-luciferase reporter assays were performed 36 hr post-transfection, and the fold inhibition of Luc activity by α-amanitin was calculated. Variation between constructs in the fold induction measured for the CMV promoter-driven Renilla construct (CMV-Renilla) was used for normalization. Total cellular RNA from the same experiments was subjected to qPCR, and GAPDH served as the internal control. The fold inhibition by α-amanitin is plotted and the data are presented as mean ± SD (n = 3).

Figure 4

Simultaneous Visualization of Pol III and II Transcripts Made by the H1 Promoter (A) Four H1 promoter-based constructs with the termination signals (T6 for Pol III and pA for Pol II) are indicated. (B) Predicted transcript lengths. (C) Probing of Pol III/II transcripts on northern blot. Total cellular RNA from DNA-transfected HEK293T cells was isolated, and a fixed amount (5 μg) was subjected to northern blotting. The RNA size marker M was used for estimation of the transcript sizes. The rRNAs and tRNAs were stained with ethidium bromide and used as a loading control. The Pol III and Pol II transcripts are indicated (*minor Pol III transcripts of ∼80 nt that likely reflect termination at non-T6 signals; this becomes signal **for construct 4 due to the change in the local sequences). (D) Probing of ∼1,800-nt Pol II transcripts on northern blot. A fixed amount (15 μg) of total RNA was subjected to agarose-formaldehyde gel electrophoresis and northern blotting. The ethidium bromide staining of an RNA ladder was used for the estimation of transcript sizes.

Figure 5

The Effect of α-Amanitin on Transcription from Pol III Promoters (A) Pol II/III promoters were cloned into the P-T6-pA-Luc backbone. (B) The P-T6-pA-Luc constructs were individually transfected into HEK293T cells with or without α-amanitin (2 μg/mL) treatment. RNA extraction and northern blotting were performed as in Figure 4C. The Pol III/II transcripts are indicated. The ** signals reflect minor Pol III transcripts (see legend to Figure 4C). The results were reproduced in a second independent experiment. (C) The P-T6-Luc constructs were individually transfected into HEK293T cells with or without α-amanitin (2 μg/mL). RNA extraction and northern blotting were performed as in Figure 4D.

Figure 6

Inactivation of the TATA Box of Type 3 Pol III Promoters Abolishes Pol III Transcription, but It Stimulates Pol II Activity (A) Mutation of the TATA boxes of the three Pol III promoters was achieved by changing the TATA sequence (+) to TCGA (−). (B) Northern blot analysis was performed as described in Figure 4C. The pBS construct was used as a negative control. The ∼44-nt transcripts (black triangle) represent Pol III transcripts terminated at T6. (C) The Luc activity of the different promoter constructs with (+) or without (−) TATA box. The pBS served as a negative control. The data are shown as mean values ± SD (n = 3).

Figure 7

Mapping the Pol II Transcription Start Site on Type 3 Pol III Promoters (A) Schematic of the mRNA 5′-RACE procedure. The Cap-Clip Acid Pyrophosphatase is used to specifically hydrolyze the 5′ cap of mRNA. (B) Amplification of Pol II-specific transcripts made by the three promoters. The 5′-RACE was performed with (+) or without (−) Cap-Clip treatment. Products of the expected size are marked by a black triangle. (C) Illustration of Pol II start site usage on Pol III promoters. The Cap-Clip-dependent products from (B) were subjected to TA-cloning and Sanger sequencing. The Pol II transcription start site was determined by aligning the sequencing output with the DNA construct. The start position was related to the position of Pol III transcription initiation, which was arbitrarily defined as +1. (D) Sequences around the Pol III/II transcription start sites. Pol III start sites (solid arrow) and Pol II start sites (dotted arrow) are indicated. The TATA boxes of these Pol III promoters are underlined and the position is indicated. The relative Pol III and II strengths (right panel) were derived from the results represented in Figure 1.