Putative molecular mechanisms underlying tandem CCCH zinc finger protein mediated plant growth, stress and gene expression responses

Abstract

In animals, Tandem CCCH Zinc Finger (TZF) proteins control a variety of cellular processes via regulation of gene expression at transcriptional and post-transcriptional levels. Plant-unique TZF proteins can also affect many aspects of plant growth, development and stress responses. However, the molecular mechanisms underlying plant TZF function are unknown. The purpose of this short review is to provide an overview of genetic and molecular analyses of plant TZFs, and to speculate on their possible molecular functions.

Zinc finger genes constitute a large and diverse gene family. Based on their individual finger structure and spacing, zinc finger proteins are further divided into many different families that are usually associated with specific molecular functions. For example, C₂H₂ zinc finger motifs are involved in DNA-binding whereas RING zinc finger motifs are protein-protein interaction domains.¹ Zinc finger genes are involved in a variety of processes including RNA packaging, transcriptional activation, regulation of apoptosis, protein folding and assembly, lipid binding, etc.¹ Tandem CCCH Zinc Finger (TZF) genes in particular, belong to one of the smallest zinc finger families in mammals. The TZF motif is characterized by two CCCH zinc fingers separated by 18 amino acids.² A genome-wide analysis of zinc finger proteins in Arabidopsis identified 26 putative TZF genes.³ Among them, only 2 (At1g66810 and At1g68200) have the same zinc finger motif (Cx₈Cx₅Cx₃H-x₁₈-Cx₈Cx₅Cx₃H) present in mammalian TZFs. Surprisingly, 11 of the 26 genes were found to contain a plant-specific TZF motif variant of Cx₇₋₈Cx₅Cx₃H-x₁₆-Cx₅Cx₄Cx₃H.^3,4 Although animal TZFs are involved in post-transcriptional regulation of gene expression via RNA-binding,^5,6 the molecular function of the plant-unique TZF motif remains unclear. In this review, we provide a brief overview of the current understanding of plant TZF function based on genetic studies, biochemical analyses and a molecular modeling approach.

Genetic Analysis of Plant TZF Function

In humans, the TZF family consists of three genes: TTP, BRF1 and BRF2.⁷ Compared to mammals, plants have much larger TZF gene families. In arabidopsis, rice and soybean, there are a total of 11, 9 and 23 TZF genes, respectively. Within each of these families, the expression of each TZF gene varies widely and is affected by developmental stages and environmental cues. For example, of the 11 AtTZF genes, AtTZF4, five and six are seed- and embryo-specific. AtTZF3 is highly expressed in senescing leaves, and AtTZF2, and seven are preferentially expressed in the vasculature.⁸ The temporal and spatial expression patterns of TZFs are also evident in other plant species. In soybean, there are also three seed-specific TZF genes (Glyma06g44440, Glyma12g33320 and Glyma13g371100) as well as a set of root nodule-specific TZFs (Glyma04g05290, Glyma11g27130, Glyma02g39210).⁹

In addition to their tissue-specificity, TZF genes are also responsive to environmental cues. In Arabidopsis, AtTZF1 and two are strongly repressed by sugar¹⁰ while AtTZF4 is repressed by phytochrome-mediated light signals.¹¹ In this way, despite potential functional redundancy due to sequence similarity among TZF families, individual TZF genes are able to play distinct roles in response to both developmental and environmental cues. For instance, in Arabidopsis, seed-specific SOMNUS (AtTZF4) is involved in phytochrome-mediated seed germination,¹¹ while salt-inducible AtSZF1 (AtTZF11) and AtSZF2 (AtTZF10) are positive regulators of salt tolerance.^8,12 In cotton, GhZFP1 expression is upregulated by salt, osmotic stress and the defense signal hormone salicylic acid. Overexpression of GhZFP1 in tobacco enhances both salt tolerance and pathogen resistance.¹³ In rice, the expression of OsDOS (Oryza sativa Delay of the Onset of Senescence) is downregulated during leaf senescence, panicle development and pollination.¹⁴ RNAi knockdown of OsDOS caused accelerated leaf senescence while overexpression caused delayed senescence.¹⁴

However, when these gene specific expression patterns are overridden by ubiquitous ectopic expression during reverse genetic analysis, TZF genes lose their specificity and the resultant overexpressing phenotypes are surprisingly similar. For instance, overexpression of AtTZF1, whose transcript is not normally upregulated during osmotic stress, like AtTZF10 and 11, resulted in enhanced tolerance to cold and drought stresses.¹⁰ Similarly, despite AtTZF1 not being a seed specific gene,⁸ its overexpression caused a significant reduction in light-dependent seed germination (Pomeranz M and Jang JC, unpublished results). Conversely, ectopic expression of seed-specific AtTZF4, five or six genes in whole Arabidopsis plants resulted in the production of compact and stress-tolerant phenotypes similar to that of overexpression of AtTZF1 (Bogamuwa S and Jang JC, unpublished data). These results indicate that plant TZFs share certain degrees of functional redundancy when misexpressed and thus are likely to have a conserved basic molecular function.

Possible Molecular Mechanisms Underlying Plant TZF Function

AtTZF1 and RNA binding.

In animals, the molecular function of TZF genes has been well studied. It is well known that human TZF (hTTP) can bind to class II AU-rich elements (AREs) in the 3′UTR of target genes via its central TZF motif,⁵ and recruit ARE-mRNAs to P-bodies (PBs) for silencing via RNA decay and translational repression.^5,15 hTTP induces rapid turnover of its target mRNA by recruiting and activating RNA degradation enzymes.¹⁶ In Arabidopsis, early results of in vitro assays suggested that like hTTP, AtTZF1 (At2g25900) could bind RNA in a zinc dependent manner.⁴ Moreover, mutations of cysteine residues in either one of the AtTZF1 CCCH motifs abolished RNA binding, suggesting that the interaction was mediated via its central TZF motif (Pomeranz M and Jang JC, unpublished data). This again is similar to what has been found in hTTP.¹⁷ Nevertheless, attempts to determine if AtTZF1 could bind known hTTP target class II ARE sequences were negative.⁴ Thus, while the RNA targets of other Arabidopsis CCCH zinc finger proteins have been found, such as pre-mRNA bound by HUA1,^18,19 and polyadenylation signals bound by AtCPSF30,²⁰ no known nucleic acid target been identified so far for TZFs.

AtTZF1 zinc finger structure.

To better understand the ARE binding characteristics of animal and plant TZF motifs, an in silico approach was undertaken. We used the Swiss-Model tool (http://swissmodel.expasy.org),^21,22 and the mouse TTP (TIS11d) crystal structure as a template,²³ to create a computer-generated model of the central TZF region of AtTZF1 and the ARE nonamer 5′-UUA UUU AUU-3′. Using Deepview 4.0,²⁴ model viewing software, we were able to analyze the AtTZF1 predicted structure and compare it to the crystallized structure of TIS11d. The Swiss Model algorithm was effective in aligning the AtTZF1 and TIS11d backbone protein structures including the positions of CCCH residues important for coordinating Zn²⁺ (Fig. 1A–E). TIS11d's high affinity binding to RNA is largely mediated through (U8-Tyr170-U9), (U6-Phe176-A7), (U4-Tyr208-U5) and (U2-Phe214-A3) aromatic stacking²³ in which each listed Phe or Tyr aromatic side chain is sandwiched between two adjacent aromatic RNA residues (Fig. 1B and see arrows).²³ These aromatic residues are also conserved in all six CCCH fingers of the aforementioned HUA1 (Fig. 2 and highlighted in pink). While AtTZF1 also contains conserved (U6-Phe152-A7) and (U2-Phe184-A3) stacking, it lacks the interaction for U8-U9 and U4-U5 nucleotide pairs, due to the replacement of Tyr170→Arg146 and Tyr208→Arg179 (Fig. 1A and C, TIS11d residues in white and AtTZF1 residues in blue). The loss of these interactions is significant as mutations of these amino acids in hTTP to non-aromatic residues completely abolishes ARE binding.¹⁷ However, while these differences support the lack of AtTZF1 binding to the class II ARE nonamer,⁴ they do not exclude the possibility of ATZF1 binding to other RNA elements. For example, despite the replacement of similar stacking amino acids Tyr→Lys, Tyr→Glu and Tyr→Asn in each one of its three CCCH zinc fingers (Fig. 2 and highlighted in pink), AtCPSF30 can still bind RNA (although it does require an additional N-terminal motif for high affinity binding).²⁰

Figure 1.

Comparative structural analysis of TZF-ARE interaction in AtTZF1 and TIS11d. AtTZF1-ARE model was generated by using TIS11D-ARE crystal structure (1RGO1) as a template²⁴ via Swiss-Model tool (www.swissmodel.expasy.org).²¹,²² Visualization was conducted using Deepview 4.0 software.²⁴ (A) Merged model of AtTZF1-ARE and TIS11d-ARE stacking interactions. Ribbon structure of TIS11d and AtTZF1 are shown in red and green, respectively, with nonameric 5′-UUA UUU AUU-3′ RNA backbone labeled in pink. TIS11d's high affinity RNA binding is mediated through stacking interactions (RNA side chains labeled in orange) at (U8-Tyr170-U9), (U6-Phe176-A7), (U4-Tyr208-U5) and (U2-Phe214-A3). Aromatic amino acid side chains involved in TIS11d binding are colored in white and their AtTZF1 counterparts are colored in blue. White labels denote critical residues from TIS11d/AtTZF1 (e.g., Tyr170/Arg146). (B) Stand alone model of TIS11d-ARE interaction showing stacking interactions at (U8-Tyr170-U9), (U6-Phe176-A7), (U4-Tyr208-U5) and (U2-Phe214-A3). (C) Stand alone model of AtTZF1-ARE interaction showing that Phe152 and Phe184 stacking interactions are conserved in AtTZF1 (green check marks). However, due to Tyr170 to Arg146 and Tyr208 to Arg179 changes U8/U9 and U4/U5 stacking interactions are abolished (red cross marks). (D) Space filling model of the six amino acids at the N-terminus of each zinc finger that form RNA binding surface in AtTZF1 (HYSGTA and RYRTQP, blue) and TIS11d (RYKTEL and KYKTEL, white), respectively. While both form similar pocket structure, amino acids important for TIS11d-ARE recognition are not conserved in AtTZF1. It is unknown if AtTZF1 amino acid variants in this region could provide comparable interactions. (e) Clustalw alignment of TZF motif of TIS11d and AtTZF1. Residues important for high affinity RNA binding are highlighted in pink. Residues important for ARE recognition and binding pockets formation are highlighted in blue and grey, respectively.

Figure 2.

Protein sequence alignments of various CCCH fingers. AtTZF1 and its homologs in humans (hTTP, P26651) and in rice (OsDOS, Os01g09620) are compared with other Arabidopsis CCCH RNA binding proteins HUA1 (At3g12680) and AtCPSF30 (At1g30460). Alignment was conducted using Clustalw. All zinc fingers contain a conserved aromatic residue F or Y between C and H (pink). However, only hTTP and HUA1 proteins contain a F/Y residue between the 2^nd and 3^rd C. These residues are critical for ARE-RNA affinity.¹⁷,²⁶ AtCPSF30, though still able to bind target RNA without the 1^st aromatic residue, it cannot do so without the aid of an additional plant specific motif found at its N-terminus.²⁰ The binding target of the plant specific TZF contained in AtTZF1 and OsDOS is still unknown. ZF, zinc finger.

To further assess RNA binding specificity, we compared other residues in TIS11d important for ARE recognition and binding.^23,26 Specifically, the N-terminal lead-in sequence R/KYKTEL preceding each CCCH zinc finger forms binding pockets and makes up a significant amount of the RNA binding surface.²³ These sequences, though not conserved in AtTZF1, form very similar pockets that could also accommodate nucleic acid binding (Fig. 1D). However, while the structure of the pockets appears to be similar, several key amino acids involved in hydrogen bonding to the ARE sequence are not conserved in AtTZF1 (Fig. 1E and light blue). Together, these results suggest that although AtTZF1 is unable to bind 5′-UUA UUU AUU-3′ nonamer, it may still bind other RNA targets.

AtTZF1 and DNA binding.

In animals, TZF proteins can traffic between the nucleus and cytoplasmic foci²⁷ and bind both DNA and RNA.⁴ Thus, in addition to their cytosolic roles in post-transcriptional regulation via RNA binding,^5,15 some animal TZFs have also been shown to have secondary nuclear roles in transcriptional regulation. For example, in mice, TIS11d can activate transcription of a luciferase reporter gene in transactivation assays in cell culture,²⁸ while in C. elegans, PIE-1 indirectly regulates transcription by affecting RNA polymerase machinery.²⁹ In rice, the non-tandem Cx₇Cx₅Cx₃H zinc finger gene OsLIC (Oryza sativa Leaf and tiller angle Increased Controller) acts as a transcriptional activator in yeast and can bind single stranded and double stranded DNA in vitro.³⁰ In Arabidopsis, PEI1 (AtTZF6) could also localize to the nucleus and bind dsDNA.³¹ In our hands, AtTZF1 could also traffic between the nucleus and cytoplasm and bind single-stranded and double-stranded DNA in vitro.⁴ Experiments using transactivation assays in yeast and maize however, detected no transcriptional activity for AtTZF1.³² Since in these experiments AtTZF1 remained largely in cytoplasm, it is possible that AtTZF1 may still have transcriptional activity but is affected by poor nuclear localization. On the other hand, it is also possible that AtTZF1, like PIE-1,^29,33 could indirectly regulate transcription through other unknown mechanisms or play entirely different roles in nuclear processes other than regulation of transcription.

TZF as a protein interaction motif in plants.

Similar to other plant TZFs, overexpression of cotton TZF protein GhZFP1 could enhance plant salt and drought tolerance. Interestingly, Response to Dehydration 21a (RD21a) and Pathogenesis Related protein 5 (PR5) proteins were identified as GhZFP1 interacting partners in a yeast two-hybrid (Y-2-H) screen.¹³ Moreover, deletion analyses revealed that the GhZFP1 TZF region was the protein interaction domain. In animals, hTTP can also mediate protein-protein interactions via its central TZF region.³⁴ In Arabidopsis, initial Y-2-H screens have also identified an interaction between AtTZF1 and another Response to Dehydration protein (Zhang L and Jang JC, unpublished results). Though it has not been confirmed if this interaction is mediated through the TZF motif, it appears that these protein-protein interactions are conserved among plant species and may be biologically significant.

Conclusions and Perspectives

In addition to RNA binding mediated functions, animal TZFs can also play other roles in DNA binding and protein-protein interactions.^28,29,34,35 In plants, the mechanisms underlying TZF functions have not been determined. The similar phenotypes found in overexpression plants suggest that genes containing the plant-unique TZF motif may play similar roles via conserved molecular mechanisms. Independent identification of Response to Dehydration proteins as an AtTZF1 and GhZFP1 interaction partner is also supportive of this hypothesis. While gel-shift binding experiments and AtTZF1 in silico structure both show that AtTZF1 does not bind the conserved Class II ARE elements targeted by animal TZFs, the similarity in forming nucleic acid binding surfaces from other residues suggests that AtTZF genes may still bind other RNA or DNA targets.

Abbreviations

ABA

abscisic acid

SA

salicylic acid

JA

jasmonic acid

GA

gibberellic acid

PB

processing body

PEG

polyethylene glycol

TZF

tandem CCCH zinc finger

hTTP

human tristetraprolin

TIS11d

mouse zinc finger protein 36

UTR

untranslated region

AREs

AU-rich elements