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 (RPC32β) that led to the demonstration of two human Pol III isoforms (Pol IIIα and Pol IIIβ). RPC32β-containing Pol IIIβ is ubiquitously expressed and essential for growth of human cells. RPC32α-containing Pol IIIα 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 RPC32α expression impedes anchorage-independent growth of HeLa cells, whereas ectopic expression of RPC32α 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.T ranscription in eukaryotes is mediated by three nuclear DNAdependent RNA polymerases (Pol I, Pol II, and Pol III) (1, 2). Pol III directs transcription of small noncoding RNAs that are involved in translation, splicing, and other cellular processes. Transcription by Pol III is directed by at least three distinct promoter types. Type 1 (5S RNA) and type 2 [tRNA, Alu RNA, and adenoviral viral-associated (VA) RNA] promoters are internal to the gene. Type 3 (U6 and 7SK RNA) promoters are located 5′ to the transcription initiation site (3). The transcription factors that directly recognize these promoters [type 1 by gene-specific TFIIIA and general initiation factor TFIIIC; type 2 by TFIIIC; and type 3 by gene-specific PSE-binding transcription factor/small nuclear RNA-activating protein complex (PTF/SNAPc)] have been well characterized and shown to recruit general initiation factor TFIIIB to their cognate promoters (reviewed in ref. 4). Overall, the multisubunit compositions of TFIIIC and TFIIIB have been conserved from yeast to human (5, 6), but two distinct isoforms of TFIIIB have been identified in human cells-one (TFIIIB-β) active in transcription of type 1 and type 2 promoters and one (TFIIIB-α) active in transcription of type 3 promoters (7). This functional difference reflects the presence of BRF1 in TFIIIB-β and of its paralogue BRF2 in TFIIIB-α (8, 9).Pol III is highly conserved from yeast to humans and composed of 17 subunits. Of these subunits, five are common to all three polymerases, two are shared by Pol I and Pol III, and five are paralogous to subunits found in Pol I and Pol II. However, five subunits are specific to Pol III without a counterpart in Pol I or Pol II (reviewed in refs. 5 and 10). Three of these five Pol III-specific subunits (RPC32, RPC39, and RPC62) form a dissociable ternary subcomplex that is specifically required for transcription initiation (11). This ternary complex...

Gene duplication and neofunctionalization: POLR3G and POLR3GL

“…We identified POLR3GL during a mass spectrometry analysis of Pol III highly purified from HeLa cells, and determined that these cells contain two forms of Pol III, one containing POLR3G and the other POLR3GL, consistent with previous results (Haurie et al 2010). We show that POLR3G and POLR3G arose from a DNAbased gene duplication, probably in a common ancestor of vertebrates, and we describe the genome-wide occupancy of these two forms of Pol III in IMR90 cells, a nontransformed and nonimmortalized human cell line, as well as in normal mouse liver and mouse hepatocarcinoma cells.…”

“…Suppression of each isoform by siRNA suggested that POLR3GL, but not POLR3G, is essential for cell survival. Moreover, ectopic expression of POLR3G, but not POLR3GL, leads to anchorage-independent growth in partially transformed human IMR90 fibroblasts (Haurie et al 2010). Together, these results suggest that POLR3G and POLR3GL carry out different functions in the cell, but what these functions may be is unclear.…”

“…The ratios of POLR3GL over POLR3G, as determined by normalized spectral abundance factor (see Methods), were 0.45 and 0.53 in the singly and doubly purified material, respectively, indicating a lower amount of POLR3GL than POLR3G in these transformed cells. POLR3G and POLR3GL proteins correspond to the RPC32-alpha and RPC32-beta proteins described by Haurie et al (2010) and are encoded by two different genes: POLR3G located on chromosome 5 and POLR3GL located on chromosome 1, respectively.…”

“…Interestingly, the two isoforms were found to be differentially expressed, with POLR3G (RPC32-alpha) decreasing during differentiation and increasing during cellular transformation relative to POLR3GL (Haurie et al 2010). Indeed, POLR3G is one of the most highly up-regulated genes in undifferentiated human stem cells relative to differentiated cells (Enver et al 2005), and decreasing its levels results in loss of pluripotency (Wong et al 2011).…”

“…Recently, an isoform of POLR3G, RPC32-beta or POLR3GL (RPC7-Like, RPC7L), encoded by a separate gene, was identified by database searches (Haurie et al 2010). Interestingly, the two isoforms were found to be differentially expressed, with POLR3G (RPC32-alpha) decreasing during differentiation and increasing during cellular transformation relative to POLR3GL (Haurie et al 2010).…”