The tandem zinc finger RNA binding domain of members of the tristetraprolin protein family

Abstract

Tristetraprolin (TTP), the prototype member of the protein family of the same name, was originally discovered as the product of a rapidly inducible gene in mouse cells. Development of a knockout (KO) mouse established that absence of the protein led to a severe inflammatory syndrome, due in part to elevated levels of tumor necrosis factor (TNF). TTP was found to bind directly and with high affinity to specific AU-rich sequences in the 3'-untranslated region of the TNF mRNA. This initial binding led to promotion of TNF mRNA decay and inhibition of its translation. Many additional TTP target mRNAs have since been identified, some of which are cytokines and chemokines involved in the inflammatory response. There are three other proteins in the mouse with similar activities and domain structures, but whose KO phenotypes are remarkably different. Moreover, proteins with similar domain structures and activities have been found throughout eukaryotes, demonstrating that this protein family arose from an ancient ancestor. The defining characteristic of this protein family is the tandem zinc finger (TZF) domain, a 64 amino acid sequence with many conserved residues that is responsible for the direct RNA binding. We discuss here many aspects of this protein domain that have been elucidated since the original discovery of TTP, including its sequence conservation throughout eukarya; its apparent continued evolution in some lineages; its functional dependence on many key conserved residues; its "interchangeability" among evolutionarily distant species; and the evidence that RNA binding is required for the physiological functions of the proteins. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Keywords: RNA binding proteins; deadenylation; mRNA decay; tristetraprolin; zinc finger proteins.

Fig.1.. Amino acid frequency at each position of the TZF domain in eukaryotes.

The search parameters for the data summarized in (A) are described in the text. Shown in the histogram are the frequencies of a given amino acid, as percentages of all amino acids at a given site, color coded as shown at the top. At the bottom of the graph is shown the amino acid sequence of the 64-amino acid TZF domain of human TTP, with two numbering systems: One is for the TZF domain itself, with residues listed from 1–64, and the other is from the protein sequence of human TTP from NP_003398.1. Indicated are the lead-in sequences to each finger, and the first and second zinc fingers. The zinc coordinating residues are in bold type. In (B) are the aligned TZF domains from human (Hs) TTP, Drosophila (Ds) Tis11, and S. pombe (Sp) Zfs1. The arrowheads on top indicate possible phosphorylation sites (Thr) within the lead-in regions, and the arrows indicate the two arginines that may contribute to the nuclear localization signal within the linker region. The zinc coordinating cysteines and histidines are in orange and blue, respectively. The aromatic amino acids that interact with the sidechains of H26 or H64, or with RNA bases, are in green. Among the three TZF domains, 68% of the residues are invariant or highly conserved, as indicated by the asterisks and colons, respectively.

Fig. 2.. Selected TZF domains from Florideophyceae and Pezizales.

In (A) are shown the TZF domain sequences from TTP family proteins from a number of red algae. The bar at the right indicates those from Florideophyceae that have an amino acid addition in the C-x8-C interval of the first zinc finger domain. In (B) are shown the TZF domains from TTP family members expressed in Pezizales (fungi), with the sequence from S. pombe shown at the top for comparison. The bar at the right indicates fungi from the Pezizales with an amino acid “missing” from the C-x8-C interval in the second zinc finger domain. The zinc coordinating cysteines and histidines are in yellow and blue, respectively. The aromatic amino acids that interact with the RNA bases are in green.

Fig. 3.. Electrostatic surface potential of the human ZFP36L2 and TTP TZF domains.

The electrostatic surface potentials (ESP) of the interaction surfaces of the (A) ZFP36L2 and (B) human TTP TZF domains are shown with the RNA oligomer (sticks). The ESPs (shown between −10 to + 10 J/K) were produced using APBS (Jurrus, Engel et al. 2018) with AMBER charges from simulations, and the figures were generated using VMD (Humphrey, Dalke et al. 1996).

Fig. 4.. Ribbon diagram of the peptide backbone of the human TTP TZF domain in complex with its 9-base binding site.

Shown are the side chains of E107 (E5 in Fig. 1A), denoted as orange spheres, in the lead-in sequence to ZF1; and R161 (R59 in Fig. 1A), shown as purple spheres, at the C+5 position of the ZF2 C-x5-C region. Dashed arrows indicate the nucleosides (sticks). Note that U5, U6, and A7 interact with E107 (E5) and R161(R59). Zinc atoms (black spheres) and the zinc coordinating residues (ball and stick) of each finger are also displayed. The N- and C-termini of the TZF domain peptides are indicated. Adapted from (Lai, Perera et al. 2014), with permission.

Fig. 5.. Paw pathology in C129R knock-in mice.

The upper panels show X-rays and micro-CT images from C116R mouse paws, with paws from their WT counterparts shown in the lower panels. A, paw X-rays from female (A) and male (B) mice at 3 months of age. C and D, paw micro-CT images from males at 4 (C) and 6 (D) months of age. Adapted from (Lai, Stumpo et al. 2018), with permission.

Fig. 6.. Solution structure models of the second zinc finger from the TZF domains from H. sapiens TTP, S. pombe Zfs1, and C. guilliermondii Zfs1.

Portions of the solution structure models are shown from the H. sapiens TTP (A), S. pombe Zfs1 (B), and C. guilliermondii Zfs1 (C) TZF domains. Shown are the backbone structures of portions of the second zinc finger from the TZF domains, including selected sidechains. The magenta spheres represent the Zn²⁺ ions. The peptide backbone ribbon and sidechain carbons are shown in a wheat color, and the atoms of the side chain residues are represented by colored spheres: white, hydrogen; red, oxygen; blue, nitrogen; yellow, sulphur. From (Wells, Hicks et al. 2015), with permission. (D) Superposition of backbone heavy atoms from H. sapiens TTP (in wheat), S. pombe Zfs1 (in cyan), and C. guilliermondii Zfs1 (in magenta) that are shown in (A), (B), and (C).

Figure 7.. Alignment, construction, and expression of TZF domain complementation strains.

(A) Alignment of TZF domains from the indicated species. Amino acids are color coded according to ClustalW pre-defined colors. The yellow highlight indicates highly conserved CCCH residues. (B) Construct used for swapping the TZF domains from various species into the site of the endogenous S. pombe Zfs1 TZF domain. (Bm = Bombyx mori (silkworm), Cg = Candida guilliermondii, Hs = Homo sapiens, Co = Chromolaena odorata, and Sp = S. pombe). In addition, a 3X Flag tag (Zfs1:Flag) was integrated into the endogenous locus with the endogenous S. pombe zfs1 3’UTR. (C) Western blot analysis of whole cell lysates isolated from the indicated strains and blotted using anti-FLAG antibodies. TZFL and TZFR indicate the amino acid residue at the beginning of the first highly conserved TZF domain lead in sequence, (R/L)YKTEL. (D) Flocculation of the indicated strains was initiated by the addition of CaCl₂ and determined using the Helm assay (see (Wells, Huang et al. 2012) for details). The percentage of cells in suspension was measured by optical density. Shown are the means of values from three independent experiments. From (Wells, Hicks et al. 2015), with permission.

Figure 8.. Expression of Zfs1 target transcripts in the TZF domain complementation strains.

Data are shown from the NanoString analysis of target transcripts in Zfs1:Flag, zfs1Δ, and the complementation strains with the S. pombe TZF domain replaced with the indicated TZF domain described in Figure 7. The NanoString assay measures the abundance of transcripts in an RNA sample, normalized to a collection of housekeeping transcripts (Fortina and Surrey 2008). (A-E) Shown are data from 5 of the 46 potential Zfs1 target transcripts that were analyzed by NanoString. Normalized counts are shown on the y-axis for the indicated transcripts as the mean values from at least four independent isolates, +/− SD. Similar data were obtained with two plant TZF domain replacements (data not shown). (F) Shown are the averages for all transcripts that were increased 2-fold or more in the zfs1Δ strain for the indicated substitution strains. These averages include 17 of the 28 transcripts from the NanoString analysis. From (Wells, Hicks et al. 2015), with permission.