The terminal loop of pri-let-7a serves as binding site for SYNCRIP. (A) In vitro RNA affinity assay of overexpressed FLAG-SYNCRIP with wild-type pri-let-7a-1 or pri-let-7a-2 or their mutants where the terminal loops were replaced with GCAA. The bound fractions were immunoblotted against FLAG-tag. (B) In vitro RNA affinity assay of FLAG-SYNCRIP with wild-type pri-let-7a-2 or pri-miR-3440b or chimeric pri-miRNA of which the stem region is from pri-miR-3440b while the loop region is from pri-let-7a-2. The bound fractions were immunoblotted against FLAG-tag. (C) RNA-EMSA assay using terminal loop sequence from pri-let-7a-2. The RNA was incubated with increasing concentration of purified recombinant his-SYNCRIP. The free RNA or bound RNA was detected by northern blot using biotin-labeled probe against the terminal loop. (D) A schematic representation of the predicted secondary structure of pri-let-7a-2. UAGAAU: red; AUCAAG: yellow; GGG: green. (E) Sequence comparison of the loop region from pri-let-7a-1 and pri-let-7a-2. (F) In vitro RNA affinity assay of FLAG-SYNCRIP with mutants from pri-let-7a-2. The bound fractions were immunoblotted against FLAG-tag. Mutated nucleotides are labeled in red. (G) In vitro RNA affinity assay of FLAG-SYNCRIP with mutants from pri-let-7a-2. The bound fractions were immunoblotted against FLAG-tag. Mutated nucleotides are labeled in red.
