Phylogenetic distribution and evolution of the linked RNA-binding and NOT1-binding domains in the tristetraprolin family of tandem CCCH zinc finger proteins

Abstract

In humans, the tristetraprolin or TTP family of CCCH tandem zinc finger (TZF) proteins comprises 3 members, encoded by the genes ZFP36, ZFP36L1, and ZFP36L2. These proteins have direct orthologues in essentially all vertebrates studied, with the exception of birds, which appear to lack a version of ZFP36. Additional family members are found in rodents, amphibians, and fish. In general, the encoded proteins contain 2 critical macromolecular interaction domains: the CCCH TZF domain, which is necessary for high-affinity binding to AU-rich elements in mRNA; and an extreme C-terminal domain that, in the case of TTP, interacts with NOT1, the scaffold of a large multi-protein complex that contains deadenylases. TTP and its related proteins act by first binding to AU-rich elements in mRNA, and then recruiting deadenylases to the mRNA, where they can processively remove the adenosine residues from the poly(A) tail. Highly conserved TZF domains have been found in unicellular eukaryotes such as yeasts, and these domains can bind AU-rich elements that resemble those bound by the mammalian proteins. However, certain fungi appear to lack proteins with intact TZF domains, and the TTP family proteins that are expressed in other fungi often lack the characteristic C-terminal NOT1 binding domain found in the mammalian proteins. For these reasons, we investigated the phylogenetic distribution of the relevant sequences in available databases. Both domains are present in family member proteins from most lineages of eukaryotes, suggesting their mutual presence in a common ancestor. However, the vertebrate type of NOT1-binding domain is missing in most fungi, and the TZF domain itself has disappeared or degenerated in recently evolved fungi. Nonetheless, both domains are present together in the proteins from several unicellular eukaryotes, including at least 1 fungus, and they seem to have remained together during the evolution of metazoans.

FIG. 1.

Interaction of human tristetraprolin (TTP) with NOT1. In (A) is shown a schematic representation of NOT1 as a scaffolding protein for a large, multiprotein complex, containing at least 2 deadenylases. This was modified from Collart and Panasenko (2012), with permission. (B) Shows the organization of the NOT1-binding domain in human TTP, and the TTP-binding domain of human NOT1, as identified by the indicated deletions and truncations of the respective fusion proteins. (C) Shows the proposed organization of the TTP mRNA binding and deadenylating activities, with TTP binding to the AU-rich region of mRNAs through its tandem zinc finger (TZF) domain, and binding to NOT1 through its C-terminal domain. NOT1 then brings into play its attached deadenylases, promoting deadenylation and accelerated destruction of the mRNA. (B, C) are taken from Fabian and others (2013), with permission.

FIG. 2.

Linked TZF domains and NOT1-binding domains of TTP family members in eukaryotes. In (A) are shown the TZF domains and their respective putative NOT1-binding domains of TTP family member proteins from various eukaryotic species in approximately descending order of complexity, starting from the 4 mouse proteins. In general, common names have been used in this figure, but specific species names can be found in the text in each case. Since this linked binding domain arrangement is much the same in all known vertebrate proteins (except for frog and fish C3H-4—see text), the mouse is the only bony vertebrate shown, with the next level that of the cartilaginous fishes, represented by the little skate. The TZF domains are shown separated by gaps consisting of various numbers of amino acids from the extreme C-terminal putative NOT1-binding domains from the same protein. The C-terminal stop codons are indicated by the dashes to the right of the protein sequence. Alignments of both domains were by ClustalW2, with its usual consensus conventions. Amino-acid display used Boxshade. In (B) is shown a tree showing the approximate time scale of evolutionary divergence of the major eukaryotic groups, from the perspective of the fungi. This was modified from Stajich and others (2009), with permission. All of the major groups at the top of the figure, the Plantae, Amoebozoa, Choanozoa, and Metazoa, contain proteins with linked TZF domains and C-terminal NOT1-binding domains. However, although most fungi contain TZF domains, as indicated, the only species that has been found to date to contain a protein with a linked TZF domain and typical NOT1-binding domains is the chytrid fungus, Spizellomyces punctatus, as indicated by the asterisk. The dashed line indicates the uncertainty about the position of the Microsporidia, as discussed in Stajich and others (2009). See the original reference for further details.

FIG. 3.

Models of TTP family member putative NOT1-binding sites associated with the NOT1 protein of the same species. These solution structure models are based on the original coordinates of Fabian and others (2013); a view of the human complex discussed in that paper is shown in (A). The other models are based on the sequences of the predicted NOT1 protein orthologues in those species, and the predicted TTP family member protein C-terminal domains, discussed in the text and in Fig. 2A (Fabian and others 2013). The initial structures were obtained by homology modeling with appropriate mutations on the X-ray crystal structure of the C-terminal segment of human TTP bound to human NOT1. These initial models were then solvated in water, followed by a series of equilibration trajectories, and were finally subjected to lengthy molecular dynamics simulations over 30 ns using standard molecular dynamics protocols at constant temperature and constant volume. The hydrophobic TTP family protein residues that are in contact with NOT1 residues are shown in yellow, and the polar and charged TTP family protein residues which are in contact with NOT1 residues are in red. Residue numbers were omitted for clarity. (A) Homo sapiens; (B) Chromolaena odorata (Christmas bush); (C) Acanthamoeba castellanii; (D) Monosiga brevicollis; (E) Spizellomyces punctatus; (F) Dictyostelium discoideum.

FIG. 4.

Alignment of C3H-4 sequences from 2 species of frogs and 5 species of fish. Protein alignments from the apparent C3H-4 orthologues from the listed species were aligned with ClustalW2, using their defaults and labeling conventions. The GenBank accession numbers from these species were as follows: NP_571014.1 (Danio rerio); CAA71245.2 (Cyprinus carpio); XP_003458454.1 (Oreochromis niloticus); XP_004574367.1(Maylandia zebra); XP_003966860.1 (Takifugu rubripes); NP_001108269.2 (Xenopus laevis); and NP_001039082.1 (Silurana tropicalis). The solid overline indicates the position of the TZF domain; the dotted over- and underlines indicate the third and fourth zinc fingers, which do not align in all species by this method. The position of the highly conserved C-terminal domain is also indicated, which we speculate may be an unusual NOT1-binding domain in this protein. See the text for further details.

FIG. 5.

Model of the hypothetical NOT1-binding site from D. rerio C3H-4 associated with NOT1 from the same species. The sequence of the postulated atypical NOT1-binding site from D. rerio is indicated in Fig. 4. The NOT1 sequence shown is from NP_001073420.1, amino acids 817–990. The solution simulation structure was constructed by the methods described in the legend to Fig. 3. The hydrophobic (in yellow) and polar (in red) TTP residues that are in contact with NOT1 residues (in gray) are shown. The residue numbers are from the D. rerio C3H-4 protein, NP_571014, as follows: L²⁹¹LL²⁹³PL²⁹⁵A²⁹⁶L²⁹⁷RL²⁹⁹Q³⁰⁰.