Localization of RNAPII and 3' end formation factor CstF subunits on C. elegans genes and operons

Abstract

Transcription termination is mechanistically coupled to pre-mRNA 3' end formation to prevent transcription much beyond the gene 3' end. C. elegans, however, engages in polycistronic transcription of operons in which 3' end formation between genes is not accompanied by termination. We have performed RNA polymerase II (RNAPII) and CstF ChIP-seq experiments to investigate at a genome-wide level how RNAPII can transcribe through multiple poly-A signals without causing termination. Our data shows that transcription proceeds in some ways as if operons were composed of multiple adjacent single genes. Total RNAPII shows a small peak at the promoter of the gene cluster and a much larger peak at 3' ends. These 3' peaks coincide with maximal phosphorylation of Ser2 within the C-terminal domain (CTD) of RNAPII and maximal localization of the 3' end formation factor CstF. This pattern occurs at all 3' ends including those at internal sites in operons where termination does not occur. Thus the normal mechanism of 3' end formation does not always result in transcription termination. Furthermore, reduction of CstF50 by RNAi did not substantially alter the pattern of CstF64, total RNAPII, or Ser2 phosphorylation at either internal or terminal 3' ends. However, CstF50 RNAi did result in a subtle reduction of CstF64 binding upstream of the site of 3' cleavage, suggesting that the CstF50/CTD interaction may facilitate bringing the 3' end machinery to the transcription complex.

Keywords: CTD phosphorylation; RNA Polymerase II; RNAPII termination; Transcription; cleavage stimulatory factor; torpedo model.

Figure 1.

Localization of RNAPII and CstF subunits along isolated C. elegans genes. (A) Individual example gene. The gene model from IGV browser is aligned at the bottom with exons shown as boxes and introns as lines. The height of the ChIP peak represents the number of reads covering each location along the gene (soc-2). H3K9ac regions with greater than 90% hybridization over the genome are indicated by a box. (B–C) Metagene of 1,691 expressed genes. Metagenes were calculated by taking genes greater than 2 kb and scaling their gene body (+500 to +2500) to the same size. Five′ ends (−500 bp to +500 bp) and 3′ ends (2500 bp to 3500 bp) were not scaled. The signal was averaged at each base pair and the individual positions averaged across 50 bp windows. An arrow indicates the transcription start site and the 3′ end by a dotted vertical line. The x-axis indicates the location along the metagene in bp. The y-axis indicates normalized reads relative to an input DNA library (see Methods). (B) RNAPII and Ser2p. (C) CstF subunits. (D) Metagene of 242 non-expressed genes based on mRNA-seq data. RNAPII (red), Ser2p (purple), CstF64 (green), CstF50 (blue) and H3K9me3 (gray) were quantitated by ChIP-seq, and H3K9ac (black) by ChIP-on-chip.

Figure 2.

Ratios of Ser2p and CstF subunits in expressed genes. The ratio of Ser2p/RNAPII metagenes (A), (B) CstF64/RNAPII (salmon) and CstF50/RNAPII (purple), (C) CstF64/Ser2p (green) and CstF50/Ser2p (blue) and (D) CstF64/CstF50 (gray).

Figure 3.

Representative snapshots of C. elegans operons from ChIP experiments. RNAPII, Ser2p, CstF64 and CstF50 are ChIP-seq experiments and H3K9ac (black) is a ChIP-on-chip experiment. Dotted lines mark genes boundaries. Transcription is from left to right. (A) A two-gene operon, CEOP1452. (B) A three-gene operon, CEOP5100. (C) An eight-gene operon, CEOP1484, which contains an internal promoter (red arrow).

Figure 4.

Localization of RNAPII and CstF subunits in expressed C. elegans operons. Metagene of operon genes divided by first gene (n = 184), internal genes (n = 111) and terminal genes (n = 705). (A) Metaoperon showing RNAPII, Ser2p and H3K9me3. (B) Metaoperon showing CstF64, CstF50 and H3K9me3.

Figure 5.

CstF64 localization to C. elegans genes and operons is altered by CstF50 knockdown. ChIP-seq experiments of CstF64 in untreated (green) and CstF50 RNAi'ed (light green) samples. (A) Metagene analysis of expressed and isolated genes based on mRNA-seq data (n = 1,691). (B) Metaoperon of first (n = 184), internal (n = 111) and terminal (n = 705) genes.