1. Volume 41, Issue 5, Pages 579-588 (March 2011)
U1 snRNA Directly Interacts with Polypyrimidine Tract-Binding Protein during Splicing Repression 
Shalini Sharma, Christophe Maris, Frédéric H.-T. Allain, Douglas L. Black 
Molecular Cell 
Volume 41, Issue 5, Pages (March 2011)
DOI: /j.molcel
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Figure 1
U1 snRNP/5′ Splice Site Interaction Is Altered during Splicing Repression
(A) Schematic maps of the wild-type and mutant N1 exon containing pre-mRNAs.
(B) CU-elements flanking the N1 exon are essential for splicing repression. In vitro splicing of the wild-type and mutant BS713 transcripts was carried out in HeLa (lanes 1–3) and WERI-1 (lanes 4–6) extract. The RNA splicing products and intermediates are shown to the right.
(C) C-src transcripts containing the N1 exon were site-specifically 32P labeled at the N1 exon 5′ splice site. The labeled wild-type and PTB binding site mutant RNAs were incubated in Buffer DG (lanes 1–3 and 10–12), HeLa extract (lanes 5–7 and 13–15), and WERI extract (lanes 7–9 and 16–18). After incubation, reactions were treated with MNase (lanes 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, and 18). Extracts were either mock treated or preincubated with oligonucleotide U11-15 prior to addition of the labeled N1 RNA. The protected fragments were extracted using PCA, ethanol precipitated, separated using urea-PAGE, and visualized by phosphorimaging. The presence of a downstream 3′ splice site did not alter the MNase protection pattern in HeLa extract (see Figure S1).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
U1 snRNP Protects a Much Longer Region of the Pre-mRNA under PTB-Dependent Repression
(A) The extent of protection of the labeled c-src RNAs was determined using oligonucleotide-mediated RNase H cleavage. Site-specifically labeled wild-type RNAs were incubated in HeLa extract (lanes 1–8) and WERI extract (lanes 9–16). After incubation, reactions were treated with MNase (lanes 2–8 and 10–16). The protected fragments were extracted using PCA and ethanol precipitated. The protected RNAs were then subject to RNase H cleavage in presence of 15 nt long oligonucleotides as indicated. After RNase H treatment, the RNAs were separated using urea-PAGE and visualized by phosphorimaging. Oligos with complimentarity offset from those shown here gave equivalent results in both HeLa and WERI extracts (see Figure S2).
(B) Sequence of the N1 exon containing pre-mRNA. Positions of DNA oligonucleotides used for RNase H cleavage are indicated above the sequence. The lines below the sequence indicate the boundaries of nuclease protection in HeLa and WERI extracts.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 PTB Interacts with the U1 snRNA during Repression
(A) Schematic outline of the method used for studying U1 snRNA/PTB interaction. The MS2 hairpin-tagged N1 exon RNAs were incubated in HeLa extract. The assembled complexes were purified using the MS2 affinity tag method. The purified complexes were exposed to UV-254 nm and immunoprecipitated using anti-PTB antibody, PTB-NT that was prebound to gamma-bind Sepharose. The immunoprecipitated complexes were then treated with SDS/Proteinase K while still bound to the gamma bind beads, and total RNA was extracted, ethanol precipitated, labeled with 32P-pCp, and visualized using urea-PAGE.
(B) Western blot analysis of the proteins from the purified complexes. Proteins from complexes purified on wild-type RNA and mutant RNAs containing mutations in the downstrem PTB binding sites were separated using SDS-PAGE and probed using anti-PTB and anti-U1C antibodies.
(C) Analysis of RNA from purified complexes. RNA from complexes assembled and purified on wild-type (lanes 2–7) and mutant RNAs (lanes 8–13) were exposed to UV-254 nm (lanes 2, 4, 5, 7, 8, 10, 11, and 13) and subject to immunoprecipitation using beads alone (lanes 2, 5, 8, and 11) or with beads containing the anti-PTB antibody, PTB-NT (lanes 3, 4, 6, 7, 9, 10, 12, and 13). In HeLa extract, no additional proteins were recruited to the region between the N1 exon 5′ splice site and the PTB binding site (see Figure S3).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Recombinant PTB Binds to U1-SL4 RNA
(A) EMSA to analyze binding of PTB to U1-SL4 RNA. Wild-type and mutant U1-SL4 RNAs at 50 nM were incubated with increasing concentrations of His-PTB (0, 0.1, 0.5, 0.75, 1.0, 2.5, and 5.0 μM). The complexes were separated on 8% native-PAGE.
(B) Dissociation constants (Kd) of the individual domains PTB RRM1 and PTB RRM2 in complex with U1-SL4 RNA (26 nt) and CU 5-mer RNA as determined by ITC at 30°C. The substrate at the lowest concentration is in the calorimeter cell. The upper panel displays the raw electrical power trace of the binding titration (baseline set at 0 μcal/s). The lower panel plots the integrated and normalized heat signal for each injection in the binding titration against the stoichiometry.
(C) On the left panel, overlay of 1H-1H TOCSY spectra of U1-SL4 RNA26 (26 nt) free (blue peaks) and bound (red peaks) to PTB RRM2 at 40°C at a ratio 1 to 1 recorded on the Bruker Avance 900 MHz spectrometer and on the right panel, secondary structure of U1-SL4 RNA26 that highlights in green the cytidine and in blue the uridines that are the most shifted upon binding of PTB RRM2.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Competition with Free U1-SL4 RNA Shifts the Interaction between the U1 snRNP and the Pre-mRNA to that Seen in the Absence of PTB
The c-src N1 exon-containing transcripts were site-specifically labeled at the N1 exon 5′ splice site. Prior to addition of the labeled transcript to HeLa and WERI extracts, the extracts were preincubated with increasing concentrations of free wild-type (lanes 1–12) and mutant (lanes 13–22) U1-SL4 RNAs at 0, 2.5, 5.0, and 10 μM. After preincubation, the site-specifically labeled transcript was added and incubation continued. The reactions were then treated with MNase. The protected fragments were extracted and visualized as described in Figure 1.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Model for N1 Exon Repression by PTB
Binding of PTB to the CU-rich elements flanking the N1 exon allow its interaction with SL4 of U1 snRNA. This prevents U1 from interacting with the complex at the downstream 3′ splice site.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions