The minigene-encoded transcript is cleaved and polyadenylated at the distal PAS.

<p><b>Panel A:</b> Scheme showing the homology among vertebrates in the last exon of the Add2 gene (UCSC browser). The A1, A23 and A4 PAS are indicated. The stop codon is indicated (“Stop”). <b>Panel B:</b> Scheme of the last exon of the mouse Add2 gene (top). The regions used for the generation of the A1-A23-A4 construct. The numbering refers to the first base of the last Add2 exon. The probe used in the Northern blots experiments is indicated. The expected size of the transcripts originating from the different PAS is shown. <b>Panel C:</b> Northern blot experiment of RNA prepared from HeLa cells transfected with the A1-A23-A4 construct. The position of the Add2 brain mRNA (lane 1), expected plasmid encoded transcripts (lane 2) and rRNA are indicated. Mouse brain RNA (mBR) was used as control.</p

Point mutations in the DSE also result in the activation of cryptic PAS.

<p><b>Panel A:</b> Scheme of the constructs used in Panel B. The DSE and USE were mutated (indicated by an asterisk) as shown in detail in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058879#s2" target="_blank">Materials and Methods</a> section. <b>Panel B:</b> Northern blot of RNA prepared from HeLa (lanes 1–4) and HEK293 (lanes 5–8) cells transfected with the constructs shown in Panel A, hybridized with the mß-Add 16ex (top panel) or the GFP probes (bottom panel). The position of the rRNAs is indicated, as well as the position of the expected RNA bands (indicated by brackets). The bar graph indicates the relative levels of expression normalized with the GFP signal (Add2/GFP ratio, mean±SD of three different experiments). <b>Panel C:</b> RACE-PCR experiment of the HeLa cells transfection to map the cleavage site of Add2 transcripts. Only the second semi-nested PCR reaction is shown. The Add2 polyadenylation sites are indicated: “A” and “D”, for the plasmid or brain mRNAs, “B” and “C” for the cryptic sites. Identical results were obtained in HEK293 cells.</p

The Add2 A4 PAS DSE is necessary even after reversion of the AGUAAA to AAUAAA.

<p><b>Panel A:</b> Scheme of the constructs used in Panel B. The hexanucleotide motif AGUAAA was optimized to the canonical AAUAAA sequence (A1-A23-A4 CHm construct). The constructs having the optimized Hm together with a deletion and point mutations in the DSE are shown (A1-A23-A4 CHm/ΔDSE and A1-A23-A4 CHm/mutDSE, respectively). <b>Panel B:</b> Northern blot of RNA prepared from HeLa (lanes 1–4) cells transfected with the constructs shown in Panel A, hybridized with the mß-Add 16ex (top panel) or the GFP probes (bottom panel). The position of the rRNAs is indicated, as well as the position of the expected RNA bands (indicated by brackets). The bar graph indicates the relative levels of expression normalized with the GFP signal (Add2/GFP ratio, mean±SD of three different experiments). <b>Panel C:</b> RACE-PCR experiment of the HeLa cells transfection to map the cleavage site of Add2 transcripts. Only the second semi-nested PCR reaction is shown. The Add2 polyadenylation sites are indicated: “A” and “D”, for the plasmid or brain mRNAs, “B” and “C” for the cryptic sites.</p

An RNA lacking the DSE is not able to form RNA-protein complexes under more stringent incubation conditions.

<p><b>Panel A:</b> Scheme of the RNA probes and competitors used in Panel B. Panel B: RNA-EMSA experiment using complete HeLa nuclear extract and the RNA probes of Panel A. The different radioactive RNA probes were incubated with 2 µg of HeLa nuclear extract and 15 µg/µl of heparin to generate more stringent incubation conditions. The experiment was performed in more stringent conditions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058879#s2" target="_blank">Materials and Methods</a> section for details) than those used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058879#pone-0058879-g005" target="_blank">Figure 5</a> to make more evident the differences in complex formation efficiency.</p

Identification of proteins bound to the A4 PAS by pull down analysis.

<p><b>Panel A:</b> Pull down experiment of HeLa nuclear proteins incubated with the different RNA probes. Bound proteins were separated by SDS-PAGE and stained with colloidal Coomassie blue. The arrows indicate differentially bound proteins (label and apparent size), which were purified and identified by mass spec analysis (results are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058879#pone-0058879-t001" target="_blank">Table 1</a>). Molecular weight markers are indicated on the left. <b>Panel B:</b> Western blot analysis of the pulled down proteins TDP-43 and PTB. “Ctr” and “NE” indicates control beads and input nuclear extract, respectively. <b>Panel C:</b> Quantification of the results shown in Panel B (three independent experiments, mean±SD).</p