Two isoforms of human RNA polymerase III with specific functions in cell growth and transformation

Abstract

Transcription in eukaryotic nuclei is carried out by DNA-dependent RNA polymerases I, II, and III. Human RNA polymerase III (Pol III) transcribes small untranslated RNAs that include tRNAs, 5S RNA, U6 RNA, and some microRNAs. Increased Pol III transcription has been reported to accompany or cause cell transformation. Here we describe a Pol III subunit (RPC32beta) that led to the demonstration of two human Pol III isoforms (Pol IIIalpha and Pol IIIbeta). RPC32beta-containing Pol IIIbeta is ubiquitously expressed and essential for growth of human cells. RPC32alpha-containing Pol IIIalpha is dispensable for cell survival, with expression being restricted to undifferentiated ES cells and to tumor cells. In this regard, and most importantly, suppression of RPC32alpha expression impedes anchorage-independent growth of HeLa cells, whereas ectopic expression of RPC32alpha in IMR90 fibroblasts enhances cell transformation and dramatically changes the expression of several tumor-related mRNAs and that of a subset of Pol III RNAs. These results identify a human Pol III isoform and isoform-specific functions in the regulation of cell growth and transformation.

Fig. 1.

RPC32α and RPC32β assemble into distinct isoforms of human Pol III active in transcription of class III genes. (A) SDS/PAGE (4–20%) analysis of affinity-purified human Pol IIIα and IIIβ preparations. Purification of FLAG-HA-Myc (FHM)-RPC32α-associated Pol IIIα in the presence of 0.5% Nonidet P-40 and either 100 (lane1) or 300 mM KCl (lane 2). Purification of FHM-RPC32β-associated Pol IIIβ in the presence of 0.5% Nonidet P-40 and either 100 (lane 4) or 300 mM KCl (lane 5). The migration of FHM-RPC32α (lanes 1 and 2) or FHM-RPC32β (lanes 4 and 5) is indicated by an asterisk. Lane 3: Molecular weight standards with indicated masses. (B) SDS/PAGE (4–20%) analysis of purified Pol III transcription factors. Lane 1: 10 ng recombinant human (rh) BDP1 (TFIIIB150; ref 9). Lane 2: 12 ng rhBRF1 and 8 ng rhTBP. Lane 3: 50 ng rhPC4. Lane 4: 3 μL affinity-purified TFIIIC (Flag-TFIIIC110 cell line). (C) Transcription of the VA1 gene in a system reconstituted from highly purified transcription factors (shown in B). Lanes 2–6: 1 μL TFIIIC, 150 ng rhPC4, 6 ng rhBDP1 (TFIIIB150), 4 ng rhTBP, and 6 ng rhBRF1. Lanes 3 and 4: 10 and 20 ng Pol IIIα (A, lane 2). Lanes 5 and 6: 10 and 20 ng of Pol IIIβ (A, lane 5). Lane 1: 10 μg HeLa S3 nuclear extract.

Fig. 2.

RPC32α mRNA expression is down-regulated during human stem cell differentiation and up-regulated during cell transformation. (A) Relative mRNA levels determined by RT-qPCR (Materials and Methods) before induction of differentiation and at specific time points during 3 weeks of differentiation of human H1 ES cells. mRNAs analyzed are indicated below the respective diagrams. (B) Dot blot analysis (Materials and Methods) of RPC32α and RPC32β mRNA levels in 73 different human tissues or cell lines. Representation of RNAs from different tissues is shown in Table S1. (C) Schematic representation of relative mRNA levels of RPC32α (Left) or RPC32β (Right) in human IMR90 fibroblasts (lanes 1 and 7) and IMR90 fibroblasts that stably express the proteins indicated at the top: E6 and ras (lanes 2 and 8), E6 and st (lanes 3 and 9), E7 and st (lanes 4 and 10), E6/E7/st (lanes 5 and 11), or E6/E7/st and TERT (lanes 6 and 12). 6, HPV E6; 7, HPV E7; st, SV40 small t; TERT, catalytic subunit of telomerase. Relative mRNA levels determined by RT-qPCR (Materials and Methods) are depicted on the left. (D) Western blot analysis of nuclear extracts derived from IMR90 cells (lane 1) or derivatives of IMR90 cells expressing E6 and ras (lane 2), E6 and st (lane 3), E7 and st (lane 4), E6/E7/st (lane 5), or E6/E7/st/TERT (lane 6). The same blot was probed with anti-RPC39 antibodies (Top) or affinity-purified anti-RPC32α antibodies (Bottom; the specificity of the anti-RPC32α antibodies is shown in Fig. S8 A and B).

Fig. 3.

siRNA-mediated suppression of RPC32α impedes colony formation of HeLa cells in soft-agar assays. (A) Western blot. HeLa cells (BN51) that stably express FLAG-RPC53/neo (11) were transfected with pSuper-si32α-2/puro, and individual clones were selected after addition of 2 μg/mL puromycin. Nuclear extracts were used for affinity purification of Pol III (via FLAG-RPC53), and the eluates were analyzed with anti-RPC39 or anti-RPC32α antibodies. Purifications from 500 μg nuclear extract of FLAG-RPC53/neo HeLa cells (lane 1), FLAG-RPC53-si32α-2 clone 4 (lane 2), or clone 5 (lane 3) HeLa cells. (B) Soft agar assay, in the presence of 2 μg/mL puromycin, of HeLa cells stably expressing FHM-RPC53/puro (lane 1), FHM-RPC32α/puro (lane 2), FLAG-RPC53/neo (lanes 3 and 4), and RPC32α siRNA 32α-2 (lane 3, clone 4 and lane 4, clone 5). The numbers of colonies formed are indicated to the left.

Fig. 4.

RPC32α contributes to transformation of IMR90 fibroblasts and changes the expression of several transformation-associated Pol II genes. (A) Soft-agar assay of human IMR90 fibroblasts (lane 1) and IMR90 fibroblasts that stably express the proteins indicated at the bottom (lanes 2–5). The numbers of colonies formed are indicated on the left. (B) Atlas Cancer 1.2 microarray (Clontech). Membranes were incubated with radioactively labeled cDNAs from IMR90 cells expressing E6 and E7 (Left), E6, E7, and RPC32α (Middle), or E6, E7, and RPC32β (Right). Table S2 summarizes quantitative results obtained by using the Image Quant program. Some of the regulated RNAs are encircled (S100A4: lower dot in the red oval; circles: RFC40/green; ezrin/orange; rac1/yellow; prefoldin/light blue; KLF6/turquoise. (C) Western blot. Twenty micrograms of nuclear extract, derived from IMR90 fibroblasts (lane 1) or IMR90 fibroblasts expressing E6 and E7 (lane 2), E6, E7, and RPC32β (lane 3), or E6, E7, and RPC32α (lanes 4 and 5; two distinct clones) were separated by SDS-10% PAGE, transferred to nitrocellulose membranes, and probed with the antibodies indicated on the left. (D) RT–qPCR. Expression of individual RNAs was determined in IMR90 fibroblasts expressing E6 and E7 (blue bars), E6, E7, and RPC32α (purple bars), or E6, E7, and RPC32β (light yellow bars). The following Pol III-transcribed genes were analyzed: Vault 1 RNA (lane1); tRNA^Glu (lane 2); Initiator tRNA_i^Met (lane 3); BC200 RNA (lane 4); U6 RNA (lane 5); 5S RNA (lane 6); and 7SK RNA (lane 7). The height of the bars indicates the relative expression level compared with the expression in IMR90, E6, and E7, which was set as 1.