1. Volume 13, Issue 1, Pages 91-100 (January 2004)
Autoregulation of Polypyrimidine Tract Binding Protein by Alternative Splicing Leading to Nonsense-Mediated Decay 
Matthew C Wollerton, Clare Gooding, Eric J Wagner, Mariano A Garcia-Blanco, Christopher W.J Smith 
Molecular Cell 
Volume 13, Issue 1, Pages (January 2004)
DOI: /S (03)

2. Figure 1 Skipping of PTB Exon 11
(A) Schematic structure of the PTB gene in the region of exons 8–12. Skipping of exon 9 produces PTB while inclusion of PTB 9 via alternative 3′ splice sites produces PTB 4 or 2 (minor isoform). Primers used for RT-PCR detection of exon 9 and 11 alternative splicing are shown by the half arrows.
(B) Sequence surrounding exon 11. The 34 nt exon is shown in bold. The predicted branch point is 351 nt upstream and is shown in bold large font with the surrounding consensus sequence in bold. Potential PTB binding TCTT motifs are underlined.
(C) RT-PCR of PTB mRNA using primers in exons 8 and 12. M: PTB markers prepared by PCR from recombinant PTB4, PTB2, PTB4tr, PTB2tr, PTB1, and PTB1tr (descending size). 1: no template negative control; 2: freshly isolated rat aorta smooth muscle cells; 3: rat aorta smooth muscle cells after 4 days in culture; 4: rat PAC-1 cells.
(D) Schematic structure of full-length PTB and predicted PTBtr. Alternatively spliced exon 9 and 11 coded segments are shown in black. The short frame-shifted segment of exon 12 at the C terminus of PTBtr is shaded gray.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2 PTBtr mRNA Is Degraded by Nonsense-Mediated Decay
(A) Western blot using antibodies against hUpf1/rent1 and β-actin. Lanes 1 and 2 contain equivalent amount of total cell protein 2 days after treatment with siRNAs against hUpf1/rent1 (lane 1) or negative control C2 siRNA (lane 2). Lanes 3–7 contain progressive 2-fold dilutions of protein from lane 2 to allow estimation of the degree of knockdown.
(B) RT-PCR of HeLa cell PTB after treatment of cells with siRNA targeted against Upf1 or with a control (C2) siRNA duplex. (Left panel) PCR primers in exons 10 and 12 were used to analyze splicing at various time points after the second administration of siRNA. Lanes marked “−” had no siRNA transfection. (Right panel) PCR primers in PTB exons 8 and 12 were used to analyze alternative splicing of both exons 9 and 12 of PTB 2 days after siRNA treatment. While the detectable levels of exon 11 skipping were increased 10- to 20-fold by Upf1 knockdown, exon 9 skipping/inclusion was unaffected.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3
Splicing of a PTB Exon 11 Reporter In Vitro Is Activated by 3′ Truncation
(A) Schematic structure of the PYP reporter transcript. The 5′ exon is rat α-tropomyosin exon 2 (138 nt), the 5′ part of the intron (thick black line, 78 nt) is derived from human β-globin intron 2. The remaining 535 nt included 424 nt upstream of PTB exon 11, the 34 nt exon, and 77 nt of downstream sequence. The predicted branch point and pyrimidine tract of PTB exon 11 are denoted by the gray circle and rectangle, respectively, while potential PTB binding UCUU motifs are denoted by the vertical black lines. The PYP Δ RNA was terminated 75 nt downstream of the predicted branch point.
(B) Splicing time course after incubation of full-length (FL) and 3′ truncated (Δ) PYP RNAs in HeLa nuclear extract for the indicated times. Lane 11 (3D) shows a debranched 3 hr sample of Δ RNA, indicating the position of the linearized lariat.
(C) UV crosslinking of in vitro splicing reactions from (B). Lanes 1 and 2 show total UV crosslinking of FL and Δ RNAs in HeLa nuclear extract. Lanes 3–8 show immunoprecipitation with monoclonal antibodies against PTB (lanes 3 and 4), U2AF65 (lanes 5 and 6), control mAb (raver1, lanes 7 and 8).
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4 PTB Protein Inhibits Splicing of PTB Exon 11 In Vitro
(A) Western blot showing depletion of PTB from HeLa nuclear extract. Lanes 1–3 show 12.5, 25, and 50 ng of recombinant PTB1. Lanes 10–12, 2 μl of whole nuclear extract (N), mock-depleted (M), and PTB-depleted (D) extract. Lanes 4–9 show serial dilutions of whole nuclear extract equivalent to depletion of PTB by 50%, 75%, 80%, 85%, 90%, or 100% (compared to lane 10).
(B) Recombinant proteins used for addback experiments. Shown is 1 μg of each recombinant protein. Note that PTBtr protein concentration was normalized by the quantity of the upper of the two bands so that the effective concentration of PTB1tr used in subsequent experiments is a conservative estimate. M, molecular weight markers (97, 67, 58, 56, 43, 36, and 29 kDa).
(C) In vitro splicing of PYP FL RNA. Splicing reactions were carried out for 3 hr in whole nuclear extract (N, lanes 2), mock-depleted extract (M, lanes 3), or PTB-depleted extract (D, lanes 4–9). Recombinant PTB1, PTB4, PTB1tr, and UNR were added to depleted extracts to the following final concentrations: 0.037, 0.111, 0.333, 1, and 3 μM, lanes 5–9, respectively. Lane 7 contains a quantity of recombinant PTB that restores the starting undepleted concentration. Lanes marked “−” are unprocessed transcript.
(D) UV crosslinking of PYP FL RNA to whole nuclear extract (N, lane 1), mock-depleted extract (M, lane 2), and PTB-depleted extract (D, lanes 3–6). Lanes 4–6 contain 3 μM recombinant PTB1, PTB4, or PTB1tr. Lane M: radiolabeled size markers (116, 97, 67, 58, 56, 43, 36, and 29 kDa). Lanes 7–9 show UV crosslinking of 3 μM PTB1, PTB4, or PTB1tr to PYP FL RNA in the absence of extract.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5 PTB Inhibits PTB Exon 11 In Vivo
(A) Schematic structure of the pG minigene reporter. It contains the 34 nt exon 11 with 538 nt and 189 nt of the upstream and downstream PTB introns (thin black lines), respectively. The flanking heterologous 5′ splice site (17 nt) and 3′ splice site (109 nt) regions are denoted by thick black lines. Note that because the stop codons associated with either splicing pattern are in the 3′ terminal exon, both splicing patterns are predicted to lead to NMD-insensitive products.
(B) RT-PCR analysis of PTB exon 11 splicing in the pG construct (left panel) and endogenous PTB (right panel) after treatment with control siRNAs (C2) or Upf1 siRNAs.
(C) Cotransfection of pG reporter with expression constructs for PTB1, PTB4, raver1, and CELF4. Lane marked “−” is a control lane with cotransfection of pGem4Z DNA.
(D) RNAi knockdown of HeLa PTB using siRNA duplexes against PTB (P1, P2) or control siRNA (C2). Western blot using antibodies against PTB and β-actin. Lanes 1–3 contain equivalent amount of total cell protein. Lanes 4–7 contain progressive 2-fold dilutions of protein from lane 3 to allow estimation of degree of knockdown by P1 and P2 siRNAs. P1 knocks down PTB by ∼90% and P2 by ∼75%.
(E) RT-PCR of pG splicing after treatment with siRNAs targeting PTB: P1 (lanes 1–6), P2 (lanes 7–12), and control siRNA (C2, lanes 13–18). Lanes 2–6, 8–12, and 14–18 show the effects of increasing amounts of a cotransfected PTB1 expression vector (0.012, 0.037, 0.111, 0.333, and 1 μg). Values under lanes 1, 7, and 13 represent mean ± SD for three independent repeats. While PTB knockdown increased exon 11 inclusion (compare lanes 1, 7, and 13), overexpression of PTB led to increased exon 11 skipping (lanes 1–6, 7–12, and 13–18).
Molecular Cell  , DOI: ( /S (03) )