Structural basis of pre-let-7 miRNA recognition by the zinc knuckles of pluripotency factor Lin28

Abstract

Lin28 inhibits the biogenesis of let-7 miRNAs through a direct interaction with the terminal loop of pre-let-7. This interaction requires the zinc-knuckle domains of Lin28. We show that the zinc knuckle domains of Lin28 are sufficient to provide binding selectivity for pre-let-7 miRNAs and present the NMR structure of human Lin28 zinc knuckles bound to the short sequence 5'-AGGAGAU-3'. The structure reveals that each zinc knuckle recognizes an AG dinucleotide separated by a single nucleotide spacer. This defines a new 5'-NGNNG-3' consensus motif that explains how Lin28 selectively recognizes pre-let-7 family members. Binding assays in cell lysates and functional assays in cultured cells demonstrate that the interactions observed in the solution structure also occur between the full-length protein and members of the pre-let-7 family. The consensus sequence explains several seemingly disparate previously published observations on the binding properties of Lin28.

An RNA-binding Protein, Lin28, Recognizes and Remodels G-quartets in the MicroRNAs (miRNAs) and mRNAs It Regulates

“…Recent atomic structures of Lin28 in complex with fragments of pre-let-7 loop sequences did not detect G4s (33,36). However, the structure of the free RNA may not be the same as that bound to Lin28 because our data suggest that Lin28 unwinds G4 quartets.…”

“…Lin28 induces a conformational change in its RNA targets (32,36,37,56). We hypothesized that the Lin28-induced RNA rearrangement might remodel the RNA G4 structure.…”

“…In vitro binding assays suggested that specific guanine (26,32,33), adenosine (26), uracil (34), and cytosine (35) nucleotides affect Lin28 binding. The crystal structures of Lin28 bound to pre-let-7 loop oligonucleotides suggested that the CSD and ZnF domains form critical contacts with guanine and adenine bases in distinct G-rich regions in the loops of pre-let-7s (33,36,37). In particular the ZnF domain was proposed to bind to a GGAG motif in the loop sequence of prelet-7 miRNAs.…”

“…In particular the ZnF domain was proposed to bind to a GGAG motif in the loop sequence of prelet-7 miRNAs. However, the NMR structure of isolated ZnFs complexed with AGGAGAU suggested that an exact GGAG sequence is not required (36). The Lin28 CSD appears to have little sequence specificity because it forms crystals with d(T) 6 and r(U) 6 (37).…”

A novel mechanism of LIN-28 regulation oflet-7microRNA expression revealed by in vivo HITS-CLIP inC. elegans

“…Regardless of the regulatory mechanism, LIN28 binds sequences in the terminal loop of pri-or pre-let-7 in mammals (Newman et al 2008;Piskounova et al 2008;Heo et al 2009). X-ray crystallography and NMR studies show that the Zinc-finger domains of LIN28 recognize a "GGAG" motif in a sequence-specific manner, through hydrogen bonds between the amino acid residues and the edge of the bases (Nam et al 2011;Loughlin et al 2012). Interestingly, not all copies of the 15 human let-7 genes contain the GGAG motif in their loop and this motif is also absent in most invertebrate let-7s.…”