Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Mechanistic studies of a small-molecule modulator of SMN2 splicing

doi: 10.1073/pnas.1800260115

Figure Lengend Snippet: (A) In vitro SHAPE experiment with NAI and a 140-nt-long RNA template containing exon 7. 1, DMSO; 2, SMN-C2 (50 μM); 3, SMN-C2 (5 μM); 4, lacking of NAI; 5∼8, ladders generated by addition of ddATP, ddTTP, ddCTP, and ddGTP during primer extension. PAGE was carried out on a TBE–urea sequencing gel at 60 W for 3 h. Red asterisks indicate increased band intensity with 50 μM SMN-C2. See SI Appendix for the full sequence of the RNA template. (B) In vitro and in-cell SHAPE-directed modeling of exon 7 and adjacent regions. For in vitro RNA model, SHAPE stabilizing cassette (orange) and nucleotides 1∼19 (blue) are shown in sketch. For in-cell RNA model, nucleotide numbering is aligned with in vitro SHAPE template. Nucleotides 1∼18 and 120∼140 were omitted. Significant reactivity changes are indicated in red and green asterisks for in vitro and in-cell SHAPE, respectively. The secondary structures that were previously named TSL1 and TSL2 (17) are enclosed in blue boxes. (C) Differential in-cell SHAPE reactivity in SMN2 minigene-transfected 293T cells for 10 μM SMN-C2 and DMSO in TSL1. SHAPE reactivity in single-nucleotide resolution and its SD were calculated by ShapeMapper software (32). Green asterisks indicate significant SHAPE reactivity change induced by 10 μM SMN-C2.

Article Snippet: Published online 2018 Apr 30. doi: 10.1073/pnas.1800260115 PMCID: PMC5960314 PMID: 29712837 Biochemistry Mechanistic studies of a small-molecule modulator of SMN2 splicing Jingxin Wang , a Peter G. Schultz , a, b, c, 1 and Kristen A. Johnson a, 1 Jingxin Wang a California Institute for Biomedical Research, La Jolla, CA, 92037; Find articles by Jingxin Wang Peter G. Schultz a California Institute for Biomedical Research, La Jolla, CA, 92037; b Department of Chemistry, The Scripps Research Institute, La Jolla, CA, 92037; c Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, 92037 Find articles by Peter G. Schultz Kristen A. Johnson a California Institute for Biomedical Research, La Jolla, CA, 92037; Find articles by Kristen A. Johnson Author information Copyright and License information Disclaimer a California Institute for Biomedical Research, La Jolla, CA, 92037; b Department of Chemistry, The Scripps Research Institute, La Jolla, CA, 92037; c Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, 92037 1 To whom correspondence may be addressed.

Techniques: In Vitro, Generated, Sequencing, Transfection, Software

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Mechanistic studies of a small-molecule modulator of SMN2 splicing

doi: 10.1073/pnas.1800260115

Figure Lengend Snippet: (A) Biotin–streptavidin pull-down with SMN-C2-BD (2 μM)–cross-linked cell lysate visualized by Western blots with anti-FUBP1 and anti-hnRNP A1 antibody. The 20 or 80 μM SMN-C3 was used as a competitor. RNase A (10 µg/mL)/T1 (25 U/mL) mix was used to digest the endogenous RNA. The quantification of bands in Western blots was by the ratio of pull-down signal over 1% input signal in the same blot (n = 2). (B) Cellular thermal shift assay (CETSA) with THP-1 cells and a serial dilution of SMN-C3. The dose–response was observed optimal at 76 °C. FUBP1 that remained in the supernatant was visualized by Western blot with anti-FUBP1 antibody, and the bands were integrated by ImageStudio (n = 2). (C) Fluorescence polarization with SMN-C2 (200 nM), purified recombinant FUBP1 (20 μM) or hnRNP A1 (20 µM) and a 15-mer oligo-4 (used in Fig. 2C) or a 120-nt RNA that contains exon 7 (used in Fig. 3B). (D) EMSA with 10 pmol of 3′-biotin–labeled RNA (500 nt containing exon 7) and a serial dilution of SMN-C3. The gel was visualized by Northern blot with streptavidin–HRP (n = 2). (E) Dose–response of SMN-C3 (24 h) for end-point RT-PCR of FUBP1/KHSRP dual knockdown in SMN2 minigene-transfected 293T cells compared with a random siRNA control. FL SMN and Δ7 SMN were amplified by PCR with minigene vector-specific primers and resolved in a denaturing TBE–urea PAGE.

Article Snippet: Published online 2018 Apr 30. doi: 10.1073/pnas.1800260115 PMCID: PMC5960314 PMID: 29712837 Biochemistry Mechanistic studies of a small-molecule modulator of SMN2 splicing Jingxin Wang , a Peter G. Schultz , a, b, c, 1 and Kristen A. Johnson a, 1 Jingxin Wang a California Institute for Biomedical Research, La Jolla, CA, 92037; Find articles by Jingxin Wang Peter G. Schultz a California Institute for Biomedical Research, La Jolla, CA, 92037; b Department of Chemistry, The Scripps Research Institute, La Jolla, CA, 92037; c Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, 92037 Find articles by Peter G. Schultz Kristen A. Johnson a California Institute for Biomedical Research, La Jolla, CA, 92037; Find articles by Kristen A. Johnson Author information Copyright and License information Disclaimer a California Institute for Biomedical Research, La Jolla, CA, 92037; b Department of Chemistry, The Scripps Research Institute, La Jolla, CA, 92037; c Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, 92037 1 To whom correspondence may be addressed.

Techniques: Western Blot, Thermal Shift Assay, Serial Dilution, Fluorescence, Purification, Recombinant, Labeling, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Knockdown, Transfection, Control, Amplification, Plasmid Preparation