ZBP1 recognition of beta-actin zipcode induces RNA looping

Abstract

ZBP1 (zipcode-binding protein 1) was originally discovered as a trans-acting factor for the "zipcode" in the 3' untranslated region (UTR) of the beta-actin mRNA that is important for its localization and translational regulation. Subsequently, ZBP1 has been found to be a multifunctional regulator of RNA metabolism that controls aspects of localization, stability, and translation for many mRNAs. To reveal how ZBP1 recognizes its RNA targets, we biochemically characterized the interaction between ZBP1 and the beta-actin zipcode. The third and fourth KH (hnRNP K homology) domains of ZBP1 specifically recognize a bipartite RNA element located within the first 28 nucleotides of the zipcode. The spacing between the RNA sequences is consistent with the structure of IMP1 KH34, the human ortholog of ZBP1, that we solved by X-ray crystallography. The tandem KH domains are arranged in an intramolecular anti-parallel pseudodimer conformation with the canonical RNA-binding surfaces at opposite ends of the molecule. This orientation of the KH domains requires that the RNA backbone must undergo an approximately 180 degrees change in direction in order for both KH domains to contact the RNA simultaneously. The RNA looping induced by ZBP1 binding provides a mechanism for specific recognition and may facilitate the assembly of post-transcriptional regulatory complexes by remodeling the bound transcript.

Figure 1.

The KH34 didomain of ZBP1 is responsible for recognition of zipcode[1–54] RNA. (A) Schematic diagram of ZBP1 showing conserved didomain organization. (B) Representative EMSA results for full-length ZBP1, RRM12, KH12, and KH34 binding to zipcode[1–54] RNA. The filled triangle represents a 1:1 serial dilution of recombinant protein. Free RNA (*) and RNA–protein complexes (**) are labeled. (C) Quantification of the fraction of RNA bound in EMSA data for ZBP1 and KH34 were fit to the Hill equation to measure the K_{d, app} and Hill coefficient.

Figure 2.

Binding site for ZBP1 KH34 is within the first 28 nt of the zipcode. (A) Representative EMSA results for ZBP1 KH34 binding to zipcode[1–28] RNA with fit of data to the Hill equation. (B) Representative stoichiometry binding assay for ZBP1 KH34 binding to zipcode[1–28] with fit to quadratic model of saturable ligand binding. (C–E) Representative EMSA results for ZBP1 KH34 binding to zipcode[29–54], zipcode[5–44], and zipcode[1–21] RNAs. The filled triangle represents a 1:1 serial dilution or 3:1 serial dilution (for stoichiometry experiment) of recombinant protein.

Figure 3.

Selection for ZBP1 KH34 binding to doped library identifies two RNA elements required for recognition. (A) Sequence of zipcode[1–28] that was used to generate a degenerate RNA library that was designed to be 85% wild type. EMSA of ZBP1 KH34 binding to RNA pool that resulted from three rounds of selection. (B,C) Mutation of either conserved RNA element reduces affinity for ZBP1 KH34. (D) One clone isolated from the degenerate selection that harbors seven mutations binds as well as the wild-type zipcode[1–28] RNA. The filled triangle represents a 1:1 serial dilution of recombinant protein.

Figure 4.

Structure of IMP1 KH34 pseudodimer. (A) KH3 (red) and KH4 (blue) with N and C termini and secondary structures labeled. The anti-parallel arrangement of KH3 and KH4 places the putative RNA-binding surfaces on opposite ends of the molecule. (B) Detailed view of intramolecular interface. The pseudodimer is stabilized by the packing of several hydrophobic residues as well as hydrogen bonds and electrostatic interactions.

Figure 5.

Model of RNA binding by IMP1 KH34 and spacing between bound RNA sequences. (A) The putative RNA-binding surfaces of KH3 (red) and KH4 (blue) are shown with modeled RNA tetranucleotide (magenta). Anti-parallel arrangement of KH domains requires RNA backbone to undergo an ∼180° change in direction to interact with both domains at the same time, which induces looping of the RNA. (B) Systematic deletion of nucleotides within zipcode[1–28]. Changes in RNA-binding affinity are shown relative to zipcode[1–28].

Figure 6.

Conserved residues that link KH3 and KH4 function in RNA binding and granule formation. (A) IMP1 KH34 and PCBP2 KH12 adopt similar structures, with the largest deviation coming from the residues that link the KH domains. (B) Sequence alignment of amino acids that connect KH3 and KH4 from ZBP1, IMP1-3, and VgRBP1 shown with ZBP1 KH12 linker. (C) Mutation of KH34 linker to residues in KH12 linker reduces RNA binding to zipcode[1–54]. The filled triangle represents a 1:1 serial dilution of recombinant protein. (D) Expression of GFP-ZBP1 with mutant KH34 linker results in a change in subcellular localization of the fusion protein from granular to diffuse cytoplasmic.

Figure 7.

Model for RNA looping induced by binding of the ZBP1 KH34 domain and the assembly of RNA–protein complexes. (A,B) Current data allows two distinct modes of RNA binding to be modeled based on which sequences within the bipartite RNA element the individual KH domains interact with. Polarity of the RNA to the KH domains is conserved in both models. (C) Looping of the transcript may form the binding sites for additional RNA-binding proteins and nucleate the assembly of larger RNPs.