LIN28 Zinc Knuckle Domain Is Required and Sufficient to Induce let-7 Oligouridylation.

LIN28 is an RNA binding protein that plays crucial roles in pluripotency, glucose metabolism, tissue regeneration, and tumorigenesis. LIN28 binds to the let-7 primary and precursor microRNAs through bipartite recognition and induces degradation of let-7 precursors (pre-let-7) by promoting oligouridylation by terminal uridylyltransferases (TUTases). Here, we report that the zinc knuckle domain (ZKD) of mouse LIN28 recruits TUT4 to initiate the oligouridylation of let-7 precursors. Our crystal structure of human LIN28 in complex with a fragment of pre-let-7f-1 determined to 2.0 Å resolution shows that the interaction between ZKD and RNA is constrained to a small cavity with a high druggability score. We demonstrate that the specific interaction between ZKD and pre-let-7 is necessary and sufficient to induce oligouridylation by recruiting the N-terminal fragment of TUT4 (NTUT4) and the formation of a stable ZKD:NTUT4:pre-let-7 ternary complex is crucial for the acquired processivity of TUT4.

Foxf1 siRNA delivery to hepatic stellate cells by DBTC lipoplex formulations ameliorates fibrosis in livers of bile duct ligated mice.

Activation of hepatic stellate cells (HSCs) is a key event in pathogenesis of liver fibrosis and represents an orchestral interplay of inhibiting and activating transcription factors like forkhead box f1 (Foxf1), being described to stimulate pro-fibrogenic genes in HSCs. Here, we evaluated a lipidbased liver-specific delivery system (DBTC) suitable to transfer Foxf1 siRNA specifically to HSCs and examined its antifibrotic potential on primary HSCs and LX-2 cells as well as in a murine model of bile duct ligation (BDL)-induced secondary cholestasis. Foxf1 silencing reduced proliferation capacity and attenuated contractility of HSCs. Systemic administration of DBTC-lipoplexes in mice was sufficient to specifically silence genes expressed in different liver cell types. Using intravital and immunofluorescence microscopy we confirmed the specific delivery of Cy3-labeled DBTC to the liver, and particularly to HSCs. Repeated treatment with DBTC-lipoplexes resulted in siRNA-mediated silencing of Foxf1 early after BDL and finally attenuated progression of the fibrotic process. Decreased HSC activation in-effect ameliorated liver injury as shown by substantial reduction of necrotic area and deposition of extracellular matrix. Our findings suggest that Foxf1 may serve as a target gene to disrupt progression of liver fibrosis and DBTC might provide a potentially feasible and effective tool for HSC-specific delivery of therapeutic RNA.