Processing of mir-144 requires its conserved terminal loop. (A) Transfection of human mir-144/451 expression constructs into HEK293T cells and assayed by Northern blotting. We previously used these to show that biogenesis of mir-451 requires Microprocessor enhancement via its neighbor mir-144. In particular, a variant deleted for the mir-144 loop (144LD-451) is highly compromised for generation of both pre-miRNAs as well as both mature miRNAs. In contrast, specific mutations of the mir-144 loop (144LM-451) support effective miR-451 biogenesis, but exhibit complete block as pre-mir-144 hairpins. (B) Schematic of wildtype and loop variants of mir-144. The terminal loop nucleotides with high conservation are circled in blue on the wildtype structure (Hs-mir-144). In loop variants LM1-LM3, the nucleotides altered are in red. (C, D) Northern blotting (C) and luciferase activity assays (D) of the mutant mir-144 constructs. Variants LM, LM1 and LM3 are defective in both assays, and according labeled in red. mir-375 is used as transfection control for Northern blotting. (E) Alignment of the mir-144 region across diverse vertebrate species illustrates the high degree of conservation of its terminal loop, especially the 3′ region adjacent to mature miR-144-3p. (F) Analysis of Danio rerio (Dr) pre-mir-144 hairpins injected into 1-cell zebrafish embryos. Mutation of Dr pre-mir-144 loop, while maintaining its structure, blocks its maturation. The terminal loop nucleotides with high conservation are circled in blue.
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