Tertiary Structural Differences in Pri-miRNA Paralogs Promote Alternative Drosha Cleavage and Expanding Target Repertoires

MicroRNAs (miRNA) biogenesis starts with Drosha cleavage, the fidelity of which is critical for the downstream processing and eventually determines miRNA target specificity. To understand how pri-miRNA sequence and structure influence where Drosha cleaves, we studied the processing of three pri-miR-9 paralogs, which encode the same mature miRNA but differ in loop and surrounding sequences. Interestingly, pri-miR-9-1 has a unique Drosha cleavage profile due to its kinked tertiary structure which is confirmed by small angle X-ray scattering (SAXS). Pri-miR-9-1, not pri-miR-9-2 or pri-miR-9-3, generates an alternative-miR-9 with a shifted seed sequence that expands the scope of its target genes. Further analysis of low grade gliomas patient samples indicates that alternative-miR-9 plays a distinct role in preventing tumor progression. Moreover, we demonstrated that the axial bending of pri-miRNAs promotes Drosha cleavage at non-canonical sites. Our findings reveal a novel neofunctionalization mechanism for pri-miRNA paralogs and provide new guidelines in shRNA design.