Journal: Nucleic Acids Research
Article Title: Muscleblind-like 1 interacts with RNA hairpins in splicing target and pathogenic RNAs
Figure Lengend Snippet: MBNL1 recognizes a RNA hairpin upstream of the Tnnt3 fetal exon. ( A ) Cleavage pattern (left) of the 5′-end labeled Tnnt3 151-nt transcript (a 5′ truncated form of the 200 nt T5.45 RNA) encompassing the fetal (F) exon 3′ splice site (110-nt of intron 8, 41-nt of F exon) obtained with use of three structure probes. Lanes are: Ci, incubation control or no probe added; Pb, lead ions (0.25, 0.5 mM); S1, S1 nuclease (1, 2 U/µl and 1 mM ZnCl 2 was present in each reaction); T1, RNase T1 (0.5, 1 U/µl); F, formamide (statistical ladder); T, guanine-specific ladder. The sequences forming the 18-nt stem-loop structure are also indicated. Also illustrated (right) is the proposed secondary structure model of the 151-nt transcript. The cleavage sites are indicated for each probe used and the figure inset shows the probe designations and cleavage intensity classification. The F exon sequence is marked in upper case and intron 8 in lower case. The positions of the G, A and U substitutions in the 18-nt stem-loop are also indicated. ( B ) Photocrosslinking analysis indicates reduced MBNL1, but not CUGBP1, binding to the Tnnt3 Δ10, gg and au mutants in contrast to wild-type RNA. Photocrosslinking analysis was performed as described in Figure 1 using the same lysates (protein loading controls shown in Figure 1 B) except only MBNL1 FL (MBNL1) protein was used. ( C ) Tnnt3 F exon skipping is impaired in the Δ10 and au mutants compared to wild type while F exon inclusion is eliminated in the gg double mutant. C2C12 cells were co-transfected with either a wild type, Δ10, gg or au point mutant splicing reporter plasmid and a protein expression plasmid for either CUGBP1mycHis or MBNL1mycHis (full-length protein only). 32 P-labeled splicing products, which included (+F) or excluded (−F) the Tnnt3 F exon (black box), were detected by RT-PCR, using primers positioned in Tnnt3 exons 8 and 9 (open boxes with arrows), followed by gel electrophoresis.
Article Snippet: Chemical and enzymatic analysis of RNA structures Transcription reactions were carried out in a 50 µl volume which contained 2 μg of each DNA template, 1 mM rNTPs, 3.3 mM guanosine, 60 U of ribonuclease inhibitor RNase Out (Invitrogen), 200 U of T7 RNA polymerase (Ambion, Austin, TX, USA), 10 mM DTT, 40 mM Tris-HCl (pH 7.9), 6 mM MgCl2 , 2 mM spermidine, 10 mM NaCl.
Techniques: Labeling, Incubation, Sequencing, Binding Assay, Mutagenesis, Transfection, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis