ZFIN Image: Shang et al., 2022, Figure 4.

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Figure Caption

Figure 4.

Reshaping of the apical loop mediates dicing of pre-mir-144. (A) Schematic of wildtype and variant mir-144 loop constructs. In the wildtype mir-144 loop (WT 144), the stable SHAPE-MaP structure is indicated as a Dicer-incompetent state. Nucleotides predicted to re-pair as a Dicer-competent substrate (with 2bp stem from the single-stranded junction to the Dicer cut site) are connected by red lines; the putative ILF3 binding site is indicated with a blue dashed oval. In the mir-144 variants, the mutated nucleotides are in red. (B) Northern blotting and (C) activity sensor assays highlight the ‘GA’ core of the ILF3 site is essential for miR-144 processing and function (144LM4). However, the apical stem structure is not required (144LM6). (D) Northern blotting and (E) activity sensor assays of reshaping mutants show that if the 2 bp re-structured stem cannot form, the resulting pre-mir-144 variants remain Dicer-incompetent (144LM7 and 144LM8). By contrast, restoration of pairing in the reshaping mutants (144LM7R and 144LM8R) fully restored pre-mir-144 dicing.

Acknowledgments

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