Transcription of mammalian messenger RNAs by a nuclear RNA polymerase of mitochondrial origin

Abstract

Transcription of eukaryotic genes is performed by three nuclear RNA polymerases, of which RNA polymerase II is thought to be solely responsible for the synthesis of messenger RNAs. Here we show that transcription of some mRNAs in humans and rodents is mediated by a previously unknown single-polypeptide nuclear RNA polymerase (spRNAP-IV). spRNAP-IV is expressed from an alternative transcript of the mitochondrial RNA polymerase gene (POLRMT). The spRNAP-IV lacks 262 amino-terminal amino acids of mitochondrial RNA polymerase, including the mitochondrial-targeting signal, and localizes to the nucleus. Transcription by spRNAP-IV is resistant to the RNA polymease II inhibitor alpha-amanitin but is sensitive to short interfering RNA specific for the POLRMT gene. The promoters for spRNAP-IV differ substantially from those used by RNA polymerase II, do not respond to transcriptional enhancers and contain a common functional sequence motif.

Figure 1

(A) Confocal microscopy of HeLa cells stained with antibodies to POLRMT peptide and TRITC anti-rabbit IgG counterstained for nuclei with Hoehst 33258. (B) Two POLRMT gene products detected by Western in HeLa (three left lanes) and ρ0 HeLa cells (two right lanes) with antibodies to POLRMT peptide. Total-cell extracts - lanes 1,4,5; mitochondrial (lane 2) and nuclear (lane 3) fractions; sample from cells expressing siRNA to Exons 3 and 17 of human POLRMT gene (lane 5); (C) Expression of mtRNAP-encoding ORF in ρ0 HeLa cells (Western); (D) Expression of siRNA to POLRMT Intron 1 in ρ0 HeLa cells (Western with antibodies to POLRMT peptide).

Figure 2

Two distinct POLRMT-specific polypeptides are encoded by alternative transcripts. (A) Immunostaining with anti-Flag antibodies of HeLa cells transduced with flag-tagged ORF for mtRNAP (left) and with flag-tagged alternative ORF encoding spRNAP-IV (right); (B) Western analysis of proteins reactive with antibodies to a POLRMT peptide following overexpression in total cell extracts of mtRNAP ORF (lanes 2 and 4), or spRNAP-IV ORF (lanes 3 and 5) in total-cell extracts (lanes 2 and 3) or in the nuclei (lanes 4 and 5); (C) Western analysis of HeLa cells transduced with spRNAP-IV Flag-tagged at the N- or C-terminus and developed with antibodies to POLRMT (left) or to Flag (M2). (D) Expression of POLRMT siRNA affects cell growth. Specified ρ0 cell lines were infected with lentivirus vector LSLG co-expressing GFP and siRNA to POLRMT Exon 3 (or to Exon 17, which produced similar result) or GFP and siRNA to luciferase, mixed with uninfected cells, and the proportion of fluorescing cells was monitored by FACS. A control sample of H1299 cells was co-infected with lentivirus vector expressing spRNAP-IV ORF (day 14 point marked by an arrow).

Figure 3

(A) Dose-dependent stimulation of ZBTB1, MGC3265 and ALDH12 transcripts in HeLa and MRC5 cells treated for 14 hours with α-amanitin (Northern); (B) Time-dependent stimulation of the transcripts following infection of HeLa cells with lentiviral vector LSLG expressing siRNAs to RNAP I or RNAP II. Inhibition of RNAP I and II mRNAs is shown on lower panels (Northern); (C) Inhibition of the transcripts four days after infection of ρ0 HeLa cells with LSLG lentivirus expressing siRNA to POLRMT (Northern), and (D) stimulation of the same transcripts after infection with a lentiviral vector expressing spRNAP-IV ORF (RT-PCR).

Figure 4

(A) Transcription from the ALDH12 and ZBTB1 promoters depends on spRNAP-IV. pGL3-luc constructs bearing ALDH12 (−482 –12) and ZBTB1 (−330 -- +12) promoters were transfected into HeLa cells (48 hours). The cultures were treated with α-amanitin for 12hrs (luciferase assay); (B) Suppression of ALDH12 promoter by siRNA to POLRMT. 24hrs after infection of HeLa cells with siRNA expressing LSLG constructs the cells were transfected with pGL3-ALDH12 (−482 –12 upstream fragment) or control plasmids and assayed for luciferase in 48 hrs; (C) Chromatin immunoprecipitation with antibodies to RNAP II or POLRMT of promoter-containing plasmids transfected into HeLa cells. The plasmids contained either ALDH12 (−290 – −12) or SV40-promoters. (D) CMV-enhancer (−600 –112) does not cooperate with ALDH12 promoter (−290 –12). 48 hours after transfection of promoter-bearing pGL3-luc constructs (ALDH12 −290 – −12, mtH-strand promoter, SV40 early promoter) the cultures were treated with α-amanitin for 12hrs and assayed for luciferase.