Journal: Cells

Article Title: Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian Cells.

doi: 10.3390/cells11223529

Figure Lengend Snippet: Figure 4. A reporter assay for splicing control by designer PPR proteins. (a) Diagram of the reporter system RG-6. The bi-chromatic alternative splicing reporter transcribes RNA containing the EGFP or dsRED coding frame in the presence or absence of exon 2. (b) Sequence of the target region. The positions of the target sequences (sp1–6) for the designer PPR proteins are depicted. The exons and introns are shown by uppercase and lowercase letters, respectively. The polypyrimidine tract is highlighted using bold letters. (c) Cell image analysis for the splicing control. Merged fluorescent images of GFP and dsRED show the designer PPR proteins of the SCD, PPR2.0, or PPR3.0 scaffolds transfected into HEK293T cells with RG-6 reporter plasmids. The control experiment was performed without transfection of the PPR gene. The white bar indicates 10 µm. (d) RT-PCR analysis of the splicing variants. The extent of alternative splicing (presence or absence of exon 3; +Ex, −Ex) was analyzed using RT-PCR with forward and reverse primer sets, as shown in (a). The skipping ratio was estimated by calculating the signal intensity of [−Ex]/([+Ex] + [−Ex]).

Article Snippet: The splicing reporter assay plasmid for RG-6 was obtained from Addgene (#80167).

Techniques: Reporter Assay, Control, Alternative Splicing, Sequencing, Transfection, Reverse Transcription Polymerase Chain Reaction