Functional equivalence of an evolutionarily conserved RNA binding module

Abstract

Members of the tristetraprolin (TTP) family of proteins participate in the regulation of mRNA turnover after initially binding to AU-rich elements in target mRNAs. Related proteins from most groups of eukaryotes contain a conserved tandem zinc finger (TZF) domain consisting of two closely spaced, similar CCCH zinc fingers that form the primary RNA binding domain. There is considerable sequence variation within the TZF domains from different family members within a single organism and from different organisms, raising questions about sequence-specific effects on RNA binding and decay promotion. We hypothesized that TZF domains from evolutionarily distant species are functionally interchangeable. The single family member expressed in the fission yeast Schizosaccharomyces pombe, Zfs1, promotes the turnover of several dozen transcripts, some of which are involved in cell-cell interactions. Using knockin techniques, we replaced the TZF domain of S. pombe Zfs1 with the equivalent domains from human TTP and the single family member proteins expressed in the silkworm Bombyx mori, the pathogenic yeast Candida guilliermondii, and the plant Chromolaena odorata. We found that the TZF domains from these widely disparate species could completely substitute for the native S. pombe TZF domain, as determined by measurement of target transcript levels and the flocculation phenotype characteristic of Zfs1 deletion. Recombinant TZF domain peptides from several of these species bound to an AU-rich RNA oligonucleotide with comparably high affinity. We conclude that the TZF domains from TTP family members in these evolutionarily widely divergent species are functionally interchangeable in mRNA binding and decay.

Keywords: RNA binding protein; RNA turnover; mRNA decay; yeast genetics; zinc finger.

FIGURE 1.

Analysis of target transcripts in strains expressing Zfs1 TZF domain point mutations. A, the sequence of the first (ZF1) and second (ZF2) zinc finger from S. pombe Zfs1. The residues highlighted in gray indicate the mutations within each zinc finger. B, Western blot analysis of whole-cell lysates isolated from the indicated strains and blotted using anti-FLAG or anti-actin antisera. C—G, data from five of the 28 transcripts that were analyzed by NanoString in the WT, zfs1Δ, Zfs1 C347R, and Zfs1 C370R strains. Diagonally striped columns represent the zfs1Δ deletion strain. Shown are the means ± S.D. from at least three independent isolates. The y axis represent normalized RNA counts for the indicated transcript. H, the averages for all transcripts increased 2-fold or more in the zfs1Δ strain for the indicated strains. These averages include 14 of the 28 transcripts from the NanoString analysis.

FIGURE 2.

Design and expression of Zfs1 TZF domain complementation constructs. A, alignment of TZF domains from the indicated species. The numbering corresponds to the position within the TZF domain. Sequences were aligned by ClustalW2, and colors were assigned by ClustalW2 on the basis of their physicochemical properties. The conserved CCCH is highlighted in yellow. Asterisks indicate the amino acid identity at that site, colons indicate a conserved substitution, and dots indicate a semiconserved substitution at that site. B, schematic of the construct used to substitute the TZF domains from various species into the endogenous S. pombe locus. In addition, a 3× FLAG tag was integrated into the endogenous locus of S. pombe Zfs1. Bm, B. mori; Cg, C. guilliermondii; Co, C. odorata; Hs, H. sapiens; Sp, S. pombe). C, Western blot analysis of whole-cell lysates isolated from the indicated strains and blotted using anti-FLAG or anti-actin antisera. TZF L1 and TZF L1R indicate the amino acid residue at the beginning of the highly conserved TZF domain lead in the sequence (L/R)YKTE(P/L), where position 1 is the first amino acid in the TZF domain. D, flocculation analysis of TZF domain replacement strains. Flocculation of the indicated strains was initiated by the addition of CaCl₂ and determined using the Helm assay (13) as described previously (4). The percentages of cells in suspension were measured on the basis of optical density at 600 nm. Shown for each strain are the means of values from three independent experiments.

FIGURE 3.

Expression of Zfs1 target transcripts in the TZF domain complementation strains. Shown is a NanoString analysis of target transcripts in the S. pombe TZF domain Leu-1, zfs1Δ, and the complementation strains in which the S. pombe TZF domain was replaced with the indicated TZF domain. L1/L1R indicates the amino acid residue at the beginning of the TZF domain lead-in sequence (L/R)YKTE(P/L). Diagonally striped columns represent zfs1Δ, and dotted columns represent the L1R mutants. The y axes represent normalized RNA counts for the indicated transcript. Bm, B. mori; Cg, C. guilliermondii; Hs, H. sapiens; Sp, S. pombe. A—E, five of the 28 transcripts that were analyzed by NanoString. The y axes indicate normalized counts for the indicated transcripts, which are shown as the mean ± S.D. from at least four independent isolates. F, averages for all transcripts 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.

FIGURE 4.

Expression of Zfs1 target transcripts in the plant TZF domain substitution strain. Shown are the results of the NanoString analysis of target transcripts in the S. pombe TZF Leu-1, zfs1Δ, and C. odorata TZF L1 strains as described in the legend for Fig. 3. Sp TZF L1 indicates the wild-type S. pombe TZF domain sequence, and Co TZF L1 indicates the wild-type TZF domain sequence from C. odorata. Diagonally striped columns represent zfs1Δ. The y axes represent normalized RNA counts for the indicated transcript. A—E, shown are five of the 28 transcripts that were analyzed by NanoString. Shown are the means ± S.D. from at least four independent isolates. F, averages for all transcripts increased 2-fold or more in the zfs1Δ strain for the indicated strains. These averages include 14 of the 28 transcripts from the NanoString analysis.

FIGURE 5.

Measurement of Zfs1 TZF domain binding to RNA using fluorescence anisotropy. A, binding reactions contained the 13-base fluorescein-labeled RNA target (5′-FL-UUUUAUUUAUUUU-3′) or a polyU control (5′-FL-UUUUUUUUUUUUU-3′). Various concentrations of recombinantly expressed, purified peptide containing the TZF domain of Zfs1 from S. pombe (Sp Zfs1 TZF) or the purified peptide containing the Sp Zfs1 C370R mutant were added to the indicated probe, and fluorescence intensity was monitored. A nonlinear regression algorithm in PRISM was used to calculate TZF domain-dependent changes in anisotropy. B, binding reactions were performed as described in A using various concentrations of the analogous peptide containing the Sp Zfs1 TZF L1R mutant as well as fusion peptides containing the H. sapiens TTP TZF R1:MBP fusion peptide and the full-length C. guilliermondii Zfs1 L1:MBP fusion protein. A nonlinear regression algorithm in PRISM was used to calculate protein-dependent changes in anisotropy.

FIGURE 6.

Construction and expression of full-length Zfs1 replacement strains. A, schematic of the construct used to substitute the full-length ORFs from the TTP family member proteins from the indicated species into the endogenous S. pombe locus. In addition, a 3× FLAG tag was integrated into the endogenous locus of the S. pombe Zfs1 protein in-frame at with the C terminus. Cg, C. guilliermondii; Mm, M. musculus; So, S. octosporus; Sp, S. pombe. B, Western blot analyses of whole-cell lysates isolated from the indicated strains and blotted using anti-FLAG or anti-actin antibodies. The lysate from the M. musculus TTP strain was diluted 1:10 to obtain comparable expression levels to the proteins from the other species. C, flocculation analysis of full-length replacement strains. Flocculation of the indicated strains was performed as described in the legend for Fig. 2. Shown are the means of values from three independent experiments.

FIGURE 7.

Expression of Zfs1 target transcripts in the Sp Zfs1 replacement with So Zfs1. Shown are the NanoString analyses of target transcripts in strains containing S. pombe Zfs1, zfs1Δ, and S. octosporus Zfs1, in which the entire Zfs1 ORF from S. pombe was replaced with the corresponding ORF from S. octosporus (So Zfs1). Diagonally striped columns represent zfs1Δ. The y axes represent normalized RNA counts for the indicated transcript. A–E, five of the 28 transcripts that were analyzed by NanoString. Shown for the indicated transcripts are the means ± S.D. from at least four independent isolates. F, shown are the averages for all transcripts increased 2-fold or more in the zfs1Δ strain for the indicated strains. These averages include 14 of the 28 transcripts from the NanoString analysis.

FIGURE 8.

Expression of Zfs1 target transcripts in the Sp Zfs1 full-length replacement strains. Shown are the NanoString analyses of target transcripts in S. pombe Zfs1, zfs1Δ, and the full-length replacement strains, in which the ORF of S. pombe Zfs1 was replaced with the indicated ORFs. Diagonally striped columns represent zfs1Δ. The y axes represent normalized RNA counts for the indicated transcript. Cg, C. guilliermondii; Mm, M. musculus; Sp, S. pombe. A–E, five of the 28 transcripts that were analyzed by NanoString. Shown for the indicated transcripts are the means ± S.D. from at least four independent isolates. F, averages for all transcripts increased 2-fold or more in the zfs1Δ strain for the indicated strains. These averages include 18 of the 28 transcripts from the NanoString analysis.

FIGURE 9.

Comparison of portions of the second zinc fingers from H. sapiens TTP, S. pombe Zfs1, and C. guilliermondii Zfs1. Portions of the solution structure models are shown from H. sapiens TTP (A), S. pombe Zfs1 (B), and C. guilliermondii Zfs1 (C) TZF domains. Shown are the backbone structures of a portion of the second zinc finger from the TZF domains, including selected side chains. The indicated residues are thought to form hydrophobic patches that help fold the terminal histidine into position to facilitate both zinc and RNA binding. The magenta spheres represent the Zn²⁺ ions. The peptide backbone ribbon and side chain 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, sulfur. See the text for further details.