Exon Tethering in Transcription by RNA Polymerase II Michael J. Dye, Natalia Gromak, Nick J. Proudfoot  Molecular Cell  Volume 21, Issue 6, Pages 849-859 (March 2006) DOI: 10.1016/j.molcel.2006.01.032 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 CoTC Sequences in β-Globin Exons 2 and 3 (A) Diagram of the β, βE2-CoTC, and βE3-CoTC constructs. The gene structure of the β-globin gene is represented by shaded boxes (exons), and introns 1 (∼120 bp) and 2 (∼850 bp) are labeled. The parallel bars in intron 2 indicate that this intron is not drawn to scale. Labeled arrows indicate the position of primers used in RT-PCR analysis. (B) RPA of β and βE2-CoTC transcripts. In the diagram above the data panel, pA indicates the position of the poly(A) site and starred lines indicate the riboprobes used. The VA RPA signal (also in [E]) derives from the cotransfected VA plasmid and controls for transfection efficiency and RNA recovery (Dye and Proudfoot, 1999). (C) RT-PCR analysis of β and βE2-CoTC transcripts. (D) RPA of β and βE2-CoTC transcripts. The riboprobe is represented by the starred line in the diagram above the data panel. Diagrams of protection products are shown to the left of the data panel. (E) RPA of β and βE3-CoTC transcripts. Molecular Cell 2006 21, 849-859DOI: (10.1016/j.molcel.2006.01.032) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 CoTC Sequences in β-Globin Intron 2 (A) Diagram of intron 2 and exons 2 and 3 of the β, βIns, and βIn-CoTC constructs. Labeled arrows indicate the primers used in RT-PCR analysis. (B) S1 nuclease analysis of β, βIns, and βIn-CoTC transcripts. The end-labeled probe used in this experiment is shown in the diagram above the data panel. The VA signal is as in Figure 1. (C) RT-PCR analysis of β, βIns, and βIn-CoTC transcripts. The identities of the PCR products are indicated in the diagrams below the data panel. M, 100 bp DNA ladder. (D) NRO and hybrid selection NRO analysis of βIn-CoTCsel. In the diagram of the βIn-CoTCsel gene construct (not to scale), the arrow indicates the start site of transcription. The filled ovals represent elongating RNA Pol II. Characters in bold type indicate the position of NRO probes. Probe M (M13 DNA) controls for background hybridization. The position of the biotinylated probe (BIO probe) is indicated by the box with tail. (E) Incubation of the T7 transcript of the βIn-CoTCsel intron 2 in nuclear extract. Molecular Cell 2006 21, 849-859DOI: (10.1016/j.molcel.2006.01.032) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Hammerhead Ribozyme Cleavage in the β-Globin Gene (A) Top, diagram of the 52 nt hammerhead ribozyme (RZ). Bottom, diagram of the β, βE2-RZ, and βE2-RZm constructs. Labeled arrows indicate the primers used for RT-PCR analysis. (B) RT-PCR analysis of RZ-directed transcript cleavage in exon 2. The diagrams to the right of the data panels indicate the structure of the PCR products. M, 100 bp DNA ladder. (C) Diagrams of intron 2 and flanking exons 2 and 3 the β, βIn-RZ, and βIn-RZm constructs. Labeled arrows indicate the primers used for RT-PCR analysis. (D) S1 nuclease analysis of β and βIn-RZ transcripts. The end-labeled probe used in this experiment is shown in the diagram above the data panel. VA signal is as in Figure 1. (E) RT-PCR analysis of β, βIn-RZ (RZ), and βIn-RZm (RZm) transcripts. The structure of the PCR products is indicated in the diagrams below the data panel. M, 100 bp DNA ladder. Molecular Cell 2006 21, 849-859DOI: (10.1016/j.molcel.2006.01.032) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Further Analysis of β, βIn-RZ, and βIn-RZm Transcripts (A) RT-PCR analysis of chromatin-associated (pellet) and nucleoplasmic (supernatant) βIn-RZ and βIn-RZm transcripts. In the diagram of β-RZ(m), labeled arrows indicate the primers used for RT-PCR analysis. The structures of the PCR products are indicated in the diagrams to the right of the data panel. M, 100 bp DNA ladder. (B) Analysis of splicing pathway of β and βIn-RZ transcripts. Top, diagrams of HindIII restriction site tagged β and βIn-RZ constructs. Middle, diagrams of the PCR products that would be generated by cis- or trans-splicing of tagged β and βIn-RZ transcripts. Bottom, RT-PCR analysis and HindIII restriction digestion of tagged β and βIn-RZ transcripts. M, 470 bp DNA marker. Molecular Cell 2006 21, 849-859DOI: (10.1016/j.molcel.2006.01.032) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Exon Tethering Model (A) Tethering of pre-mRNA to RNA Pol II. In the diagram (not to scale), the black line indicates the intron 2 DNA template. The curved line represents the nascent transcript. Gray boxes with black outline represent tethered exons. The rhombus represents splice factors associated with the intron 2 splice donor site. The scissors represent CoTC or RZ cleavage of the nascent intron transcript. Association of CBC with splice factors and the transcription complex (Görnemann et al., 2005) is not shown for illustrative reasons. (B) Cotranscriptional splicing. The black hexagon represents splice factors associated with the intron 2 splice acceptor site. The thick dashed line represents interaction between 5′ and 3′ splice factors. Molecular Cell 2006 21, 849-859DOI: (10.1016/j.molcel.2006.01.032) Copyright © 2006 Elsevier Inc. Terms and Conditions