PAN–ENE likely affects 3′-end formation. (A) Schematic diagram of the RPA. Probe complementarity does not extend into the Δ4 deletion, so that the same-sized fragments are produced for all constructs tested. (B) Results from an RPA with RNA from cells transfected with the WT or Δ4 construct, or untransfected (no TXN) cells. The Probe lane shows 1% of the total. The values under each lane are percent cleavage and the ratio of the cleaved to read-through signals normalized to WT. The latter values are shown with standard deviations in Figure 4B. (C) RNA sequence of the 3′-end formation elements of the polyadenylated (pA) and hammerhead ribozyme (Hh) constructs. The sequence shown begins with PAN nt 1046, with common nucleotides depicted in bold face and cleavage sites indicated by arrows. Important cis-acting sequences are underlined: the polyadenylation hexanucleotide, downstream GU-rich regions, and the entire hammerhead ribozyme. Vector sequence is in lowercase letters. (D) Northern blot of RNAs from cells transfected with the indicated constructs. Quantitation was performed by normalization to a control probe that hybridizes to the co-transfected Rta mRNA. The ratios are the normalized WT PAN to Δ4 signals, with the standard deviation from four independent experiments.

Definition of the 79-nt PAN–ENE core element. (A) Schematic showing the Δ4 and the Δ4 subdeletion constructs, with boxed areas representing deletions. The Δ4a–d constructs contain 57–58 nt deletions (Figure 1), while the Δ4c1–d5 constructs contain 12–14 nt deletions. (B) Quantitation of data from RPAs with the Δ4 deletion series. RPAs (Figure 2) were quantitated by setting the cleaved-to-read-through ratio of WT transcripts to 1.0 for each independent experiment; each deletion construct was compared to the WT value. The data are the average of three independent experiments, with the error bars indicating standard deviation. (C) The sequence of the 79 nt core PAN–ENE. Vertical lines demarcate the Δ4c1–d2 regions. Horizontal lines separate the three sequence domains of the element.

Multiple copies of the PAN–ENE cause accumulation of the intronless β-globin mRNA in the nucleus. Fluorescence in situ hybridization of HEK293 cells transfected with the indicated β-globin reporter constructs. Two panels are shown for each construct to illustrate the heterogeneity observed in both signal strength and localization. Since only a subset of the cells are transfected, only a fraction of the cells in each panel show hybridization signal. Signal-to-noise ratios are lower with the βΔ1,2 and βΔ1,2-79Rx4 constructs (panels E, F, I, and J), due to their relatively low levels of mRNA.

Splicing is dominant over PAN–ENE-mediated retention. Schematic diagram: Northern (A), Western (B), and in situ hybridization (C) analyses of intron-containing β-globin–PAN–ENE constructs. Analyses were performed as in Figures 5B, D, and 6. Panels c and e are independent fields showing β1-79Fx5; d and f show β1-79Rx4.

Sequences near the 3′-end of PAN are necessary for high levels of PAN RNA. (A) Schematic diagram of the PAN RNA constructs. Numbering refers to the PAN start site (+1) as defined by Zhong et al (1996). (B) Northern blot analysis of the constructs listed in (A). The control is mRNA signal from a co-transfected β-globin construct. High-molecular-weight smears representing read-through transcripts were cut off for presentation.

PAN–ENE does not alter transcription rate or transcript half-life. (A) A representative slot blot from a nuclear run-on experiment with nuclei isolated from cells transiently transfected with PAN WT or Δ4, and β-globin constructs. In the left-most panel, Rta was omitted to assess the background of the PAN probe. A probe for the cellular RNA polymerase III-transcribed 7SK RNA was also included. The probes are full-length RNA transcripts complementary to PAN, β-globin, or 7SK. (B) Relative transcription levels of the PAN WT and Δ4 constructs are graphed with the WT value set at 1.0. The data represent the average of three independent experiments with standard deviations shown. (C) RNase protection analysis of RNAs from cells transiently transfected with the PAN WT (left panel) or Δ4 (right panel) constructs. Actinomycin D was added 18–24 h post-transfection and cells were harvested at the indicated times. The probe used in the Δ4 panel had five-fold higher specific activity than the WT probe. The probe lanes show ∼1% of the total. Intervening samples between the Δ4 probe and subsequent lanes were omitted for clarity. No TXN shows RNase protection of RNA from untransfected cells. (D) Graphical representation of the decay experiments; each point is the average of three or four independent experiments. □: WT cleaved; ○: Δ4 cleaved; × : WT RT; ⋄: Δ4 RT.

PAN–ENE rescues RNA levels, but not translation of a heterologous intronless transcript. (A) Schematic representation of the β-globin constructs. The vector sequence includes the CMV promoter and bovine growth hormone polyadenylation signals (BGH pA). The light gray boxes and lines represent the β-globin exons and introns, respectively. The hatched box depicts an N-terminal Flag tag and the dark gray box (PAN–ENE) shows the site of insertion of the tested PAN fragments in forward or reverse orientation. The start (AUG) and stop (UAA) codons are also shown. (B) Northern blot analysis of the β-globin–PAN chimeric RNAs. The control is the same blot hybridized to a probe for the co-transfected Rta construct. Similar results were observed in the absence of Rta. (C) Quantitation of the results of Northern blot analysis. The β-globin signals, normalized to the control probe to correct for loading and transfection efficiency, are graphed. βΔ1,2 signal was arbitrarily set at 1.0, with other signals referenced to that signal. The relative levels shown are the averages for three or four independent experiments, with error bars representing standard deviation. (D) Western blot of the β-globin protein produced from the intronless β-globin constructs. Total protein from approximately 2 × 10⁵ cells was loaded in each lane, except lanes 2, 3, and 4 in which serial dilutions of the βΔ1 sample were loaded. The bottom panel shows the same membrane after stripping and re-probing with an antibody to HuR. Relative β-globin signals shown below are the average of two independent experiments.

Tethering of export factors leads to cytoplasmic accumulation of β-globin–ENE mRNA. (A) Schematic of export-competent mRNP formation. (B) Schematic of ENE-containing reporter constructs with MS2-binding sites. (C) In situ hybridization of β-globin reporter constructs M-βΔ1,2-79Fx5 (a, c, e, g) or βΔ1,2-M-79Fx5 (b, d, f, h) co-transfected with MS2-coat protein alone (a, b) or MS2-coat protein fused to TAP (c, d), UAP56 (e, f), or REF2-I (g, h).