Defining the RNA polymerase III transcriptome: Genome-wide localization of the RNA polymerase III transcription machinery in human cells

Abstract

Our view of the RNA polymerase III (Pol III) transcription machinery in mammalian cells arises mostly from studies of the RN5S (5S) gene, the Ad2 VAI gene, and the RNU6 (U6) gene, as paradigms for genes with type 1, 2, and 3 promoters. Recruitment of Pol III onto these genes requires prior binding of well-characterized transcription factors. Technical limitations in dealing with repeated genomic units, typically found at mammalian Pol III genes, have so far hampered genome-wide studies of the Pol III transcription machinery and transcriptome. We have localized, genome-wide, Pol III and some of its transcription factors. Our results reveal broad usage of the known Pol III transcription machinery and define a minimal Pol III transcriptome in dividing IMR90hTert fibroblasts. This transcriptome consists of some 500 actively transcribed genes including a few dozen candidate novel genes, of which we confirmed nine as Pol III transcription units by additional methods. It does not contain any of the microRNA genes previously described as transcribed by Pol III, but reveals two other microRNA genes, MIR886 (hsa-mir-886) and MIR1975 (RNY5, hY5, hsa-mir-1975), which are genuine Pol III transcription units.

Figure 1.

Three types of Pol III promoters and corresponding transcription factors. (A–C) Structures of types 1, 2, and 3 promoters, respectively, as well as the factors they recruit.

Figure 2.

Examples of POLR3D peaks with various proportions of unique and repeated tags. (A–C) UCSC Genome Browser views of the indicated genomic locations. In each case, the upper track labeled “unique” shows the peak obtained with only the unique tag component of the peak shown in the second track, labeled “all,” which contains all tags. The y-axis indicates cumulated tag weights. The track labeled “unique match coverage” shows the regions spanned by tags unique within all POLR3D peaks. Below the unique match coverage, UCSC Browser annotations for RNA genes and repeats from RepeatMasker are shown. The visual peak tracks were generated as described in the legend to Figure 3.

Figure 3.

Location of BDP1, BRF1, and POLR3D peaks on tRNA genes. (A) UCSC Genome Browser view of the indicated region of chromosome 6, which contains a number of tRNA genes. The BRF1 and BDP1 visual peak tracks were generated from an overlap profile as described in Zhang et al. (2008). The y-axis indicates cumulated tag weights. For the POLR3D visual track, the same method was used except that the tags were not extended to the actual size of the sequenced fragments to prevent any artificial tag overlaps coming from adjacent peaks reflecting adjacent RNA polymerases. Below each of the BRF1, BDP1, and POLR3D peaks, the unique match coverage is shown, indicating the regions spanned by tags unique within the BRF1, BDP1, and POLR3D peaks, respectively. (B) Zoom-in of a small region of chromosome 6. (C) The density of peak maxima for all tRNA genes displaying BDP1, BRF1, and POLR3D peaks was plotted relative to the TSSs.

Figure 4.

Location of BDP1, BRF1, and POLR3D peaks on RN5S genes. (A) UCSC Genome Browser view of the 5S cluster on chromosome 1. (B) Zoom-in on one of the RN5S gene in the cluster. The visual peak files and the markings are as in the legend of Figure 3.

Figure 5.

The MIR886 gene is transcribed by Pol III. (A) UCSC Genome Browser view of the MIR886 microRNA. The visual peak files and the markings are as in the legend of Figure 3. (B) The sequence corresponding to the POLR3D peak region (on the minus strand) is shown with putative A and B boxes indicated (the underlined nucleotides fit the A and B box consensus defined in Supplemental Fig. S1A). The likely transcription start and termination sites, as determined by POLR3D occupancy and sequence, are indicated. (C) The MIR886 gene is transcribed by Pol III in vitro. The genomic fragment carrying the MIR886 locus was immobilized on beads and used as template for in vitro transcription as described in Supplemental material. Tagetin was added in the reactions shown in lanes 2 and 4. The control template was the Ad2 VAI gene. (D) Pre-MIR886 RNA can be detected by Northern blot in IMR90hTert cells. Lanes 1 and 2 show duplicate RNA preparations. The blot was probed with a complementary oligonucleotide corresponding to regions 49–69 and 93–112 of the microRNA gene MIR886.

Figure 6.

Examples of three novel Pol III transcription units. (A) UCSC Genome Browser views of three putative new Pol III transcription units (RP3TR1, RP3TR2, and TRNAAL1). The visual peak files and the markings are as in the legend of Figure 3. (B) The sequences corresponding to the POLR3D peak regions (on the minus strand for RP3TR1 and TRNAAL1, and on the plus strand for RP3TR2) are shown. The markings on the sequence are as in the legend of Figure 3. (C) RP3TR1 and RP3TR2 are transcribed by Pol III in vitro. Genomic fragments carrying the RP3TR1 and RP3TR2 loci were immobilized on beads and used as template for in vitro transcription as described in the Supplemental material. Tagetin was added in the reactions shown in lanes 2, 4, and 6. The control template was the Ad2 VAI gene. (D) RP3TR1 and TRNAAL1 RNAs can be detected by Northern blot in IMR90hTert cells. Lanes 1–2 and 3–4 show duplicate RNA preparations. For RP3TR1, the blot was probed with complementary oligonucleotides corresponding to regions 208–237 or 303–328 of the putative gene. For TRNAAL1, the oligonucleotides corresponded to regions 23–46 and 51–71.

Figure 7.

Location of BDP1, SNAPC2, and POLR3D peaks on the TRNAU1 (tRNA-SeC) and RNU6ATAC genes. UCSC Genome Browser views showing the BDP1, SNAPC2, and POLR3D peaks, as well as the unique tag coverage indicating the regions spanned by tags unique within the BDP1, SNAPC2, and POLR3D peaks, respectively. The POLR3D, BDP1, and SNAPC2 visual peak tracks were generated as described in the legend to Figure 3. (A) TRNAU1 (tRNA-SeC) gene. (B) RNU6 snRNA gene.