A novel tandem reporter quantifies RNA polymerase II termination in mammalian cells

Abstract

Background: Making the correct choice between transcription elongation and transcription termination is essential to the function of RNA polymerase II, and fundamental to gene expression. This choice can be influenced by factors modifying the transcription complex, factors modifying chromatin, or signals mediated by the template or transcript. To aid in the study of transcription elongation and termination we have developed a transcription elongation reporter system that consists of tandem luciferase reporters flanking a test sequence of interest. The ratio of expression from the reporters provides a measure of the relative rates of successful elongation through the intervening sequence.

Methodology/principal findings: Size matched fragments containing the polyadenylation signal of the human beta-actin gene (ACTB) and the human beta-globin gene (HBB) were evaluated for transcription termination using this new ratiometric tandem reporter assay. Constructs bearing just 200 base pairs on either side of the consensus poly(A) addition site terminated 98% and 86% of transcription for ACTB and HBB sequences, respectively. The nearly 10-fold difference in read-through transcription between the two short poly(A) regions was eclipsed when additional downstream poly(A) sequence was included for each gene. Both poly(A) regions proved very effective at termination when 1100 base pairs were included, stopping 99.6% of transcription. To determine if part of the increased termination was simply due to the increased template length, we inserted several kilobases of heterologous coding sequence downstream of each poly(A) region test fragment. Unexpectedly, the additional length reduced the effectiveness of termination of HBB sequences 2-fold and of ACTB sequences 3- to 5-fold.

Conclusions/significance: The tandem construct provides a sensitive measure of transcription termination in human cells. Decreased Xrn2 or Senataxin levels produced only a modest release from termination. Our data support overlap in allosteric and torpedo mechanisms of transcription termination and suggest that efficient termination is ensured by redundancy.

Figure 1. The use of tandem reporters to measure transcription elongation.

A, A tetracycline-regulated promoter drives transcription through the tandem reporters. Self-cleaving hammerhead ribozymes cut the RNA transcript, separating both FLUC and hRLUC expressing RNA fragments from the test sequence. An internal ribosome entry sequence (IRES) enhances translation of the uncapped hRLUC expression fragment by replacing functions of the 5′ cap and untranslated region (5′ UTR). The relative ratio between the reporters measures the transcriptional impediment presented by an insert. The 5′ reporter (FLUC) is firefly luciferase and the 3′ reporter (hRLUC) is a humanized sea pansy luciferase. If the RNA polymerase terminates transcription in the inserted test sequence, the 3′ reporter will not be transcribed. B, Robust, reproducible induction of FLUC and hRLUC activities in clonal cell lines. Graphs display the mean luciferase activity per cell in Relative Light Units (RLU) for FLUC and hRLUC from clonal cell lines containing a single integrated copy of a control tandem reporter construct. Cells were cultured without (−) or with (+) the addition of doxycycline for 24 hours to induce transcription from the promoter. Extracts representing 15,000 cells were assayed for luciferase activity. The mean induction for FLUC expression was 237±29 fold, and 56±3 for hRLUC. Error bars indicate the S.E.M. for a sample number of three. C, Ribozymes self-cleave in a human cell line. Gel shows yield of RT-PCR products Transcription was induced with doxycycline and RNA was harvested after 24 hours. RT-PCR was conducted using primers designed for FLUC and hRLUC coding regions, and primers designed to span the ribozymes and the 275 bp of linker sequence between the two ribozymes. TAN-NR does not contain self-cleaving ribozymes, and all three sets of primers successfully amplify the target. β-actin primers were included as an amplification control. RT-PCR products were separated on a 1% agarose gel.

Figure 2. Actin and globin 3′ sequences provide robust transcription termination.

A, Schematic maps of DNA inserts used with the tandem vector indicating relative size. Fragments of green fluorescent protein, red fluorescent protein and chloramphenicol acetyl transferase coding regions are not full-length, and do not make functional protein. The polyadenylation regions used in the experiments are shown below, drawn to scale. The position of the poly(A) addition site is indicated by a vertical black bar. The mini actin polyadenylation insert (MnipA) would be about the width of that bar. B, Graph showing ratios of expressed hRLUC to FLUC activities for the indicated constructs in stable cell lines. Inclusion of 400 to approximately 2000 bp of sequence containing either the actin (ACT1-3) or globin (HBB1-3) poly(A) addition site effectively terminated transcription elongation. Transcription was induced with doxycycline and cells were harvested after 24 hours. Results for each cell line are expressed as a percentage of the dsred control cell line, which does not contain a polyadenylation sequence. Longer poly(A) region fragments that included a putative co-transcription cleavage element (CoTC) were included in the globin constructs, and reduced read-through expression of the downstream reporter to 1–2% of the control dsred cell line for all constructs. A minimal poly(A) signal sequence provides modest transcription termination (MnipA). The control (MnopA) contained a nearly identical sequence except for two changed bases in the hexamer part of the polyadenylation signal. The y axis values are hRLUC/FLUC expression ratios normalized to a positive cell lysate run in each plate. All of the changes are significant (p<0.05) when compared to the dsred control. Error bars indicate the S.E.M. for a sample number of three.

Figure 3. Runner sequence offers modest release from transcription termination.

A, The inclusion of 2.6 kilobases of dsred tetrameric sequence downstream (3′) to the ACT and HBB sequences provides modest release from transcription termination. Transcription was induced with doxycycline and cells harvested after 24 hours. The y-axis values are hRLUC/FLUC expression ratios normalized to a positive cell lysate run in each plate and expressed as a percentage of the dsred control cell line. All of the changes are significant (p<0.05) when compared to the dsred control. The error bars indicate the S.E.M. for a sample number of three. B, Real-time RT-PCR analysis. FLUC and hRLUC mRNA was measured by real-time RT-PCR and expressed as the relative ratio of hRLUC/FLUC mRNA. Results are shown as a percentage of the dsred control cell line. The ratio decreased as larger tracts of polyadenylation sequence are included in the tandem construct. Error bars indicate the S.E.M. for a sample number of three.

Figure 4. Xrn2 and Senataxin knockdowns provide limited release from transcription termination.

A, Western blot analysis of Xrn2 knockdown in whole cell extracts. TAN dsred control cell line was grown as indicated in the methods and transfected with a plasmid expressing an shRNA targeting Xrn2. Samples were run on duplicate gels in parallel, blotted and then probed for Xrn2 expression. Anti-Xrn2 antibody (Bethyl Laboratories) produces a doublet, shown here. The lower band disappears with shRNA targeting Xrn2, and the upper band shows reduced density. Anti-human β-actin antibody serves as a control. The analysis was performed three times, with a representative blot shown. B, Xrn2 and senataxin knockdowns each provide little release from transcription termination. Cell lines were grown as detailed in the methods. The y-axis values are hRLUC/FLUC expression ratios normalized to a positive cell lysate run in each plate. Error bars indicate the S.E.M. for a sample number of three. Significant differences (p<0.05) from the corresponding vector (pLKO) treated samples are indicated by asterisks.