The Lin28 cold-shock domain remodels pre-let-7 microRNA

Abstract

The RNA-binding protein Lin28 regulates the processing of a developmentally important group of microRNAs, the let-7 family. Lin28 blocks the biogenesis of let-7 in embryonic stem cells and thereby prevents differentiation. It was shown that both RNA-binding domains (RBDs) of this protein, the cold-shock domain (CSD) and the zinc-knuckle domain (ZKD) are indispensable for pri- or pre-let-7 binding and blocking its maturation. Here, we systematically examined the nucleic acid-binding preferences of the Lin28 RBDs and determined the crystal structure of the Lin28 CSD in the absence and presence of nucleic acids. Both RNA-binding domains bind to single-stranded nucleic acids with the ZKD mediating specific binding to a conserved GGAG motif and the CSD showing only limited sequence specificity. However, only the isolated Lin28 CSD, but not the ZKD, can bind with a reasonable affinity to pre-let-7 and thus is able to remodel the terminal loop of pre-let-7 including the Dicer cleavage site. Further mutagenesis studies reveal that the Lin28 CSD induces a conformational change in the terminal loop of pre-let-7 and thereby facilitates a subsequent specific binding of the Lin28 ZKD to the conserved GGAG motif.

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

“…Isolated studies with the ZnF domains also suggest that they partially unfold pre-let-7 targets (56). Additionally, a FRET study revealed that the CSD of Lin28 remodels the terminal loop of pre-let-7 by unwinding the upper stem region (37). We propose that the Lin28-mediated remodeling of RNA disrupts the G4 structure because in the presence of Lin28, the Lin28-binding RNAs no longer induce NMM fluorescence.…”

“…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.…”

“…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). Sequencing of Lin28-bound mRNAs suggested that Lin28 might bind to sequences containing GGAGA, AAGNNG, AAGNG(N), or (N)UGUG(N) where N is any nucleotide (31,38).…”

Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition

“…A GGAGA motif was also reported to be present in 28% of the LIN28A-HiTS-CLIP defined mRNA-binding sites and was the most significantly enriched sequence element (Wilbert et al 2012). Co-crystals of mouse or Xenopus LIN28 proteins with fragments of Xenopus prelet-7f miRNA showed that both, CSD and ZKD, interacted with single-stranded regions within the pre-let-7 loop, corresponding to a pyrimidine-rich segment and a GGAG motif, respectively (Nam et al 2011;Mayr et al 2012). These interactions were similar to those previously observed for bacterial CSDs, which bound single-stranded pyrimidine-rich sequences with very few nucleobase-specific contacts (Phadtare and Inouye 1999; Max et al 2006).…”