Identification of the SLM2-binding motifs in TTN mRNA
A. Binding probability of SLM2 to TTN intron 131 predicted from CLIP datasets using the DeepRiPe machine learning model. B. Potential SLM2-binding UWAA repeat motifs in intron 131 of TTN mRNA. C. Occurrence of UWAA motifs in the TTN exons 131–133. D. Sanger sequencing showing individual (Mut1, Mut2, Mut3, and Mut4) and double (Mut1+2 and Mut3+4) mutations of the identified UWAA motifs in intron 131 in the TTN131–133 minigene. E. and F. Analysis of TTN mRNA splicing using splice-PCR targeting exons 131–132 after transfection of HEK293 cell with the WT TTN131–133 minigene or TTN131–133 minigene with individually (E) or doubly (F) mutated UWAA motifs along with a plasmid expressing human SLM2. CLIP, crosslinking and immunoprecipitation; DeepRiPe, Deep RBP binding preference.