Terminal Exon Definition Occurs Cotranscriptionally and Promotes Termination of RNA Polymerase II  Michael J Dye, Nick J Proudfoot  Molecular Cell  Volume 3, Issue 3, Pages 371-378 (March 1999) DOI: 10.1016/S1097-2765(00)80464-5

Figure 1 NRO and Hybrid Selection NRO Analysis of HeLa Cells Transiently Transfected with Plasmids Containing the ε- and β-globin Genes (A) Upper panel, diagram of the HIVε and HIVβ plasmids. The arrow indicates the start site of transcription from the HIV promoter. The highly homologous gene structure of ε- and β-globin is represented by shaded boxes (exons); introns are labeled IVS1(∼120 bp) and IVS2(∼850 bp), respectively. The parallel bars in IVS2 indicate that this intron is not drawn to scale. p(A) indicates the position of the poly(A) site. Characters in bold type indicate the position of NRO probes. Probes 3–9 are specific to the HIVε construct. (HIVβ-specific probes are indicated in [D]). Lower panel, NRO analysis of ε-globin transcription in HeLa cells following transient transfection of HIVε. The probes used are indicated above and below the filter. Probe M is M13 DNA and is a control for background hybridization. Probe H contains histone DNA and is a positive control for Pol II transcription. The Va probe detects transcription from a cotransfected control plasmid containing the adenovirus VAI gene. The 5S probe contains 5S rDNA and measures Pol III transcription. Signals obtained from NRO analysis were normalized for background hybridization, incorporation of α32P-UTP, and transfection efficiency, using the cotransfection control plasmid pVa, before plotting on the graph in the right-hand panel. (B) Diagram showing the position of the biotinylated RNA probes used for hybrid selection of transcripts upstream (pre) and downstream (post) of the ε-globin poly(A) site. Left panel, hybrid selection NRO analysis of ε-globin transcripts using a pre–poly(A) site biotinylated selection probe. Corrected signals from both pre– and post–poly(A) site selection experiments are shown graphically in the right-hand panel. (C) NRO analysis of HeLa cells transiently transfected with the HIVβ plasmid. The probes used are indicated above and below the filter. Probes 3–10 are specific to β-globin; their position with respect to the poly(A) site is shown in (D). Other probes are as described for HIVε (A). Corrected signals are shown graphically in the right-hand panel. (D) Right upper panel, diagram showing the positions of the β-globin-specific NRO probes and the pre– and post–poly(A) site biotinylated selection probes. Left upper panel, hybrid selection NRO analysis of HeLa cells transiently transfected with the HIVβ plasmid using a pre–poly(A) site selection probe. Left lower panel, hybrid selection NRO analysis of HIVβ using a post–poly(A) site selection probe. Corrected signals from both pre– and post–poly(A) site selection experiments are shown graphically in the right-hand panel. Molecular Cell 1999 3, 371-378DOI: (10.1016/S1097-2765(00)80464-5)

Figure 2 NRO Analysis of HeLa Cells Transiently Transfected with the HIVβ, Poly(A), and Splice Site Mutant Constructs (A) Mutations that do not affect transcriptional termination. Upper panel, diagrams (not drawn to scale) of the HIVβ, HIVβSDmut, and HIVβIVS1SAmut constructs. SD and SA indicate the positions of splice donor and splice acceptor sites, respectively. Middle panel, hybridization signals, normalized for UTP content and transfection efficiency, resulting from NRO analysis of HeLa cells transiently transfected with the constructs listed above, are shown graphically using an arbitary scale. Lower panel, NRO analysis of HeLa cells transiently transfected with the HIVβIVS1SAmut plasmid. (B) Mutations that abrogate transcriptional termination. Upper panel, diagrams (not drawn to scale) of the HIVβp(A)mut, HIVβIVS2SAmut, HIVβSASDmut, and HIVβΔIVS2 constructs. Middle panel, normalized hybridization signals resulting from NRO analysis of HeLa cells transiently transfected with the constructs listed above are shown graphically using the same scale as in (A). Lower panel, NRO analysis of HeLa cells transiently transfected with the HIVβIVS2SAmut plasmid. Molecular Cell 1999 3, 371-378DOI: (10.1016/S1097-2765(00)80464-5)

Figure 3 Quantitative S1 Nuclease Analysis of Nuclear mRNA Isolated from HeLa Cells Transiently Transfected with the HIVβ, Poly(A), and Splice Acceptor Mutant Constructs (A) The 3′ β-globin probe shown in the diagram was end labeled with α32P-dATP. (B) Lanes 1–4 show protected 3′ end products following transient tranfection of HeLa cells with the constructs indicated at the top of each lane. The letters in bold type, to the right of the figure, indicate the position of the correctly 3′ end–processed β-globin mRNA and cotransfected pVa plasmid protection products. The effect of RNA processing mutations on β-globin 3′ end processing was calculated and expressed as a percentage of wild-type poly(A) site use, shown at the foot of the gel. Molecular Cell 1999 3, 371-378DOI: (10.1016/S1097-2765(00)80464-5)

Figure 4 Diagram Representing the Possible Configuration of Pol II and the Nascent RNA Transcript Located Downstream of the Poly(A) Site Pol II (in gray) and the RNA processing factors (hatched) are not drawn to scale. The gray box indicates the terminal exon, and the open box indicates the terminal intron. SA and pA denote splice acceptor and poly(A) signal (indicated by black boxes). Molecular Cell 1999 3, 371-378DOI: (10.1016/S1097-2765(00)80464-5)