PMC

β-Actin gene pause element promotes poly(A)-dependent transcriptional termination of heterologous construct in vivo. (A) Diagram of the globin/actin fusion construct, with exons shown as gray boxes, and CoTC element, substituted by the β-actin pause element, shown as a hatched box. Panels below show NRO signals for CoTC, ΔCoTC, and globin/actin fusion constructs. TE is indicated. (B) Globin/actin fusion construct, with the position of the biotinylated β4 probe just downstream of poly(A) site indicated. The panels below show transcripts selected by biotinylated β4 probes from CoTCΔpA and globin/actin ΔpA transfections. Corrected hybridization signals (with B4 taken as 1) are shown in the graph below.

Model for two types of Pol II transcriptional termination in mammalian cells. (A) CoTC-dependent termination of β-globin gene. CoTC presents a free 5′ RNA end which is recognized by the 5′→3′ exonuclease Xrn2/Rat1p. Subsequent degradation of this downstream cleavage product by Xrn2 leads to Pol II transcription termination and release of free Pol II. (B) Pause-dependent termination of β-actin gene. Due to the Pol II pausing/slowing elongation rate at the pause sequence, cleavage at the poly(A) site presents a free 5′ RNA end which is recognized by the 5′→3′ exonuclease Xrn2/Rat1p. Subsequent degradation of this downstream cleavage product by Xrn2 leads to Pol II transcription termination and release of Pol II. The double-headed arrow indicates potential interaction between Xrn2 and CTD-bound proteins.

Proximity and strength of poly(A) site are important for pause site-mediated transcriptional termination. (A) Human β-globin gene, with exons shown as gray boxes. Positions of SPA and β poly(A) site insertions are indicated; various plasmid modifications (insertion of 30 T, 30 C, and ZAM₄ elements) are also depicted on the diagram at the corresponding locations. The panels below show NRO signals for MAZ₄/U3, SPA/MAZ₄, 30T, 30C, and ZAM₄ constructs. TE is indicated. The asterisk indicates that in the case of the SPA/MAZ₄ construct, TE could not be determined. (B) Positions of the S1 probes over the β/SPA (solid line) and SPA/β (dashed line) constructs are indicated. The panel below shows S1 analysis of cytoplasmic, steady-state RNA of β/SPA and SPA/β transfections. Positions of the SPA- and β poly(A)-specific bands are indicated (based on size markers indicated).

The 5′→3′ exonuclease Xrn2 is involved in pause-dependent transcriptional termination. (A) β-Globin construct, with the position of the biotinylated β4 probe just downstream of the poly(A) site. The panels below show NRO transcripts selected by biotinylated β4 probes from CoTCΔpA, ΔCoTCΔpA, and MAZ₄ΔpA transfections. (B) Western blot analysis of protein extracts obtained from HeLa cells treated with Xrn2 or mock siRNAs. Total protein (25 or 50 μg) was analyzed using Xrn2- or actin-specific antibodies, respectively. As indicated, Xrn2 but not actin was depleted to background levels. (C) RNAi-mediated depletion of human Xrn2 protein inhibited Pol II transcriptional termination mediated by the MAZ₄ element. NRO analysis of MAZ₄ construct transfected into mock-depleted and Xrn2-depleted cells is shown. Quantitative analysis shows a 1.8- and 1.6-fold increase in Pol II density over probes A and U3, respectively, comparing Xrn2-depleted versus mock-treated cells (based on two independent experiments).

Pause-mediated transcriptional termination of endogenous β-actin gene. (A) Human β-actin gene, with exons shown as gray boxes. The positions of the probes for RT-PCR and ChIP are underlined. The sequence presented corresponds to a 380-bp pause element from the 3′ flanking region of the β-actin gene. G nucleotides are underlined. (B) Pol II ChIP analysis across β-actin gene using anti-Pol II antibody. (C) Pol II ChIP analysis across β-actin gene using primers over ex6 and regions B to D of the 3′ flanking sequence. Light-gray bars represent mock-depleted cells, and dark-gray bars are Xrn2-depleted cells. The level of Pol II density over the ex6 probe was taken as 1. (D) Real time RT-PCR analysis of the β-actin gene in mock-treated (light-gray bars) and Xrn2-depleted (dark-gray bars) cells. The level of the steady-state message was corrected relative to the level of the RNA over the ex5 probe (taken as 100%). (E) Ratios of total β-actin transcript levels detected with versus without Xrn2 depletion using real-time RT-PCR primers for exon 5, exon 6, uncleaved actin poly(A) (A), and 3′ flanking regions B, C, and D. All the quantitative ChIP and RT-PCR data were based on several independent experiments.

MAZ element promotes poly(A)-dependent transcriptional termination in vivo. (A) β-Globin construct with exons shown as gray boxes and CoTC element as hatched box. NRO probe positions are underlined, and RT-PCR probe positions are dotted lines. The panels below show NRO signals for CoTC, ΔCoTC, MAZ₄, mMAZ₄, MAZ₄ΔpA, and mMAZ₄ΔpA constructs. M (empty M13 vector) shows the background signal. The TE is shown against each NRO analysis. See Materials and Methods for details. (B) Quantitative real time RT-PCR analysis of transcription termination efficiency of CoTC, ΔCoTC, MAZ₄, and mMAZ₄ constructs. Termination efficiency is measured as the ratio of accumulation of steady-state RNA at positions A to ex2 (A/ex2). (C) S1 analysis of cytoplasmic, steady-state RNA of CoTC, ΔCoTC, MAZ₄, mMAZ₄, MAZ₄ΔpA, and mMAZ₄ΔpA transfections. Lanes P− and P+ are S1 probes (made with the CoTC plasmid) incubated without or with the S1 nuclease treatment. Note that no background bands were detected over the poly(A) site region in these lanes. Positions of the pA, pA* (cryptic), and VA (cotransfection control) bands detected in S1 analysis are indicated (based on the indicated positions of size markers). Note that read-through transcripts enhanced by mutation of the β-globin poly(A) signal were not detected in cytoplasmic RNA as they are retained in the nucleus, where they are degraded.