Volume 21, Issue 3, Pages 437-443 (February 2006) Adenylation and Exosome-Mediated Degradation of Cotranscriptionally Cleaved Pre- Messenger RNA in Human Cells  Steven West, Natalia Gromak, Christopher J. Norbury, Nicholas J. Proudfoot  Molecular Cell  Volume 21, Issue 3, Pages 437-443 (February 2006) DOI: 10.1016/j.molcel.2005.12.008 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 hscRACE Analysis of β-Globin Transcripts (A) hscRACE procedure: (1) CoTC sequence is represented by a hatched box, exon 3 is indicated by a white box, and unknown sites of CoTC are shown as lightening bolts. Enrichment of β-globin transcripts through magnetic selection of hybridized biotinylated RNA (black line and circle) upstream of the poly(A) site [p(A)] precedes release of transcripts through hybridization of DNA oligo (dotted line) followed by RNaseH cleavage. (2) Released RNA with unknown 3′ ends is ligated to form circles (3), which are then reverse transcribed with a cDNA primer (arrow) directed to amplify across the ligated junction. (4) Linear cDNA is amplified by PCR using primers (gray and black arrows) that will only amplify cDNA obtained from ligated RNA. Products were analyzed on a gel and (5) sequenced. (B) Polyacrylamide gel electophoretic analysis of hscRACE products from βΔ5–7-transfected cells. Size markers are indicated on the right-hand side. Three major products are labeled (i)–(iii). The selection of bands analyzed by cloning and sequencing is denoted by bracket. (C) CoTC RNA sequence: positions of CoTC-derived ends are indicated by arrows, where each arrow represents an individual clone. Nontemplated residues are indicated above the arrow that represents their position. CoTC hotspots are underlined. A tails observed upon PMscl100 depletion are denoted by white arrows, and their sequence is underlined in bold type. Molecular Cell 2006 21, 437-443DOI: (10.1016/j.molcel.2005.12.008) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 hscRACE Analysis of Human β-Globin CoTC (A) Western blot showing RNAi knockdown of PMscl100. The actin loading control is shown in the bottom panel, and the PMscl100 blot is shown in the top panel. (B) S1 nuclease analysis of globin mRNA with (+) or without (−) siRNA treatment. β-globin mRNA and VA RNA signals are indicated. (C) hscRACE analysis of RNA from βΔ5–7 transfected cells with (+) or without (−) siRNA treatment. PCR cycle number is indicated above the gel by the appropriate number. Bands (i)–(iii) are also indicated. (D) hscRACE analysis of βΔ5–7pAmut as in (C). PCR cycle number is indicated above the gel. Bands (i)–(iii) are again indicated. Molecular Cell 2006 21, 437-443DOI: (10.1016/j.molcel.2005.12.008) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 hscRACE Analysis of PMscl75-Depleted Cells and RNA Cleaved at the Poly(A) Site (A) RT-PCR analysis of PMscl75 knockdown. PCR products deriving from actin and PMscl75 mRNAs are shown. Presence (+) or absence (−) of PMscl75-specific siRNA is also indicated. (B) hscRACE analysis of RNA from wt (−) and PMscl75 siRNA-treated (+) cells. Bands (i)–(iii) are shown, and PCR cycle number is also indicated. (C) hscRACE analysis of RNA, from wt cells and cells treated with either PMscl75 or PMscl100 siRNAs (appropriately indicated by a +), which has been cleaved at the poly(A) site. The number of PCR cycles used is indicated. Molecular Cell 2006 21, 437-443DOI: (10.1016/j.molcel.2005.12.008) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 MSA Hybrid Selection NRO and hscRACE (A) Diagram of βAlb: β-globin exons (black) and intervening introns are shown downstream of the HIV promoter (horizontal arrow). The β-globin poly(A) site is indicated, as is the MSA 3′ flanking region insert (gray box). Single-stranded M13 probes used in NRO analysis are indicated in bold type and are underlined. (B) Hybrid selection nuclear run-on analysis of βAlb: selected (S) and unselected (US) panels are shown, and corrected hybridization signals are quantitated graphically. (C) hscRACE analysis of βAlb. Diagram shows relative positions of the biotinylated probe (black line and circle), the oligo used for RNaseH release (dotted line), and oligos used for reverse transcription (gray arrowhead) and PCR (gray and black arrowheads). Polyacrylamide gel separation of the multiple bands produced is shown on the right. The selection of bands that were cloned is indicated by a bracket. (D) Albumin CoTC sequence with positions of cloned ends indicated by black arrows. Adenylated species from wt cells (bold) and PMscl100-depleted cells (underlined) are also indicated. Molecular Cell 2006 21, 437-443DOI: (10.1016/j.molcel.2005.12.008) Copyright © 2006 Elsevier Inc. Terms and Conditions