Zinc finger proteins are ubiquitous in eukaryotic organisms and play crucial roles in a myriad of cellular processes, most notably in gene regulation. Among the diverse classes of zinc finger proteins, the CCCH family stands out due to its unique Cys-Cys-Cys-His motif and its involvement in RNA binding and post-transcriptional regulation. This study delves into the comprehensive identification and characterization of the CCCH zinc finger protein family in Arabidopsis thaliana and rice (Oryza sativa), two key model plant species. By employing bioinformatics approaches, we aim to elucidate the evolutionary relationships, structural features, and expression patterns of this important gene family, providing a foundation for understanding their diverse functions in plant biology.
Figure 1: CCCH zinc finger motif alignment. This image illustrates the conserved cysteine and histidine residues critical for the zinc-finger structure in selected CCCH proteins, highlighting the defining characteristics of the finger family.
Deciphering the CCCH Motif and Identifying Family Members
The defining feature of CCCH zinc finger proteins is the CCCH motif, characterized by the consensus sequence C-X-C-X-C-X-H, where ‘C’ represents cysteine, ‘H’ represents histidine, and ‘X’ denotes variable amino acid residues. Specifically, the canonical CCCH motif is defined as C-X6–14-C-X4–5-C-X3-H. Based on variations in the spacing between these conserved residues, the CCCH family can be further divided into subgroups. Proteins containing these motifs are considered candidate members of the CCCH zinc Finger Family.
To identify the complete repertoire of genes encoding CCCH zinc finger proteins in Arabidopsis, we conducted a proteome-wide analysis using a Perl program. This program utilized a regular expression (Cw{6,14}Cw{4,5}Cw{3}H) designed to specifically search for CCCH motifs within the Arabidopsis proteome database. Sequences matching this criteria were flagged as potential CCCH proteins.
Figure 2: CCCH motif detection program. This diagram illustrates the Perl program and regular expression used to identify CCCH motifs within protein sequences, showcasing the computational approach to discover members of the finger family.
To ensure the robustness of our findings, we employed reciprocal BLAST searches against protein databases using known CCCH protein sequences as queries. Another Perl program was used to refine these results and eliminate redundancy. Comparative analysis confirmed that the Perl program identified all proteins detected by BLAST, and thus, the Perl-derived dataset was used for subsequent analyses.
Furthermore, we employed SMART and Pfam, protein domain databases, to validate the identified CCCH protein sequences. This analysis confirmed 79 proteins with typical CCCH motifs, encoded by 68 genes in Arabidopsis. Five of these proteins showed CCCH motifs with lower confidence scores and were excluded from further analysis. However, At3G51180, despite not being detected by SMART and Pfam, was retained due to its conserved CCCH motif and presence of a sister gene in a genome duplication region. We also iteratively refined our motif search by incorporating newly discovered CCCH motif variations not initially described, expanding the total identified groups to 23.
Transcriptomic data from microarray experiments, Massively Parallel Signature Sequencing (MPSS), and EST databases corroborated the expression of all 68 identified CCCH genes, suggesting they are not pseudogenes. These genes were systematically numbered (AtC3H1 to AtC3H68) based on their chromosomal location.
Phylogenetic Analysis Reveals Evolutionary Subfamilies within the CCCH Family
To understand the evolutionary relationships within the Arabidopsis CCCH zinc finger family, we constructed a phylogenetic tree using the protein sequences of all 68 members. Due to the variability in motif number and spacing, full-length protein sequences were aligned using ClustalX and manually refined to ensure accurate phylogenetic inference. The neighbor-joining method was used to generate the phylogenetic tree, and bootstrap analysis with 1000 replicates was performed to assess the statistical reliability of the tree topology.
Figure 3: Phylogenetic tree and domain architecture of Arabidopsis CCCH family. This figure presents a comprehensive overview, including a phylogenetic tree illustrating subfamilies, gene structure with exons and introns, and protein domain architecture highlighting CCCH motifs and other conserved domains within the finger family.
While deep nodes in the phylogenetic tree showed lower bootstrap support, likely due to CCCH motif divergence, outer clades exhibited higher resolution, enabling the delineation of 11 major phylogenetic subfamilies (I to XI). This subfamily structure is consistent with observations in other plant transcription factor families. Within each subfamily, high sequence conservation is evident, indicating strong evolutionary relationships among members. Based on bootstrap support, we confidently classified the Arabidopsis CCCH family into these 11 subfamilies. Pairwise clustering with high bootstrap values (1000) identified 18 pairs of putative paralogous genes within Arabidopsis.
Conserved Motifs and Gene Structure Support Subfamily Classification
To further characterize the CCCH family, we investigated conserved motifs beyond the CCCH domain and analyzed gene structure within each subfamily. MEME, a motif discovery tool, was used to identify motifs shared among related proteins within each subfamily. SMART and Pfam were used to annotate these motifs.
As expected, proteins within the same subfamily shared common CCCH motifs. Interestingly, MEME also identified a few motifs outside of the CCCH domain. Protein motif diagrams visually confirmed the structural similarities within subfamilies. For instance, subfamily IV members contain tandem WD40 domains, and subfamily IX members possess three highly conserved tandem motifs (C-X5-H-X4-C-X3-H, C-X7–8-C-X5-C-X3-H, and C-X5-C-X4-C-X3-H). Subfamily I members are characterized by five C-X8-C-X5-C-X3-H zinc finger motifs. Furthermore, 12 CCCH proteins contain RNA-binding domains (RRM or KH motifs), suggesting their direct involvement in RNA-related processes.
Gene structure analysis revealed that intron number and gene length are also largely conserved within subfamilies, mirroring the phylogenetic relationships. Similar intron/exon structures, coupled with shared motifs, strongly support the validity of our CCCH family classification and highlight the evolutionary coherence of these subfamilies.
Genome Duplication and Divergence Shape the CCCH Family Evolution
The Arabidopsis genome’s history of polyploidy and genome-wide duplication events has significantly impacted gene family expansion. To explore the role of gene duplication in CCCH family evolution, we mapped the chromosomal location of each CCCH gene. These genes are distributed across all five Arabidopsis chromosomes, with some regions showing lower gene density.
Figure 4: Chromosomal distribution of Arabidopsis CCCH genes. This chromosome map visualizes the location of CCCH genes across the Arabidopsis genome, highlighting segmental duplication events and potential duplicated gene pairs within the finger family.
Approximately 53% of CCCH genes are located within duplicated segmental regions resulting from polyploidy events that occurred millions of years ago. We identified seven additional gene pairs with similar gene structure and zinc finger motifs that are likely also products of duplication. The high proportion of duplicated CCCH genes suggests preferential retention compared to other gene families, which is consistent with the known retention bias for genes involved in signal transduction and transcription regulation following duplication events. Segmental duplication appears to be the primary driver of CCCH gene family expansion in Arabidopsis.
To assess the evolutionary conservation of the CCCH family, we investigated the presence of homologs in moss. Preliminary BLAST searches indicated that most Arabidopsis CCCH subfamilies (I, II, III, IV, V, VI, VII, X, and XI) have homologous members in moss. Subfamily IX, however, was not detected in moss, suggesting it might be specific to higher plants or have arisen later in evolution. Further searches in angiosperm and gymnosperm databases confirmed that most Arabidopsis CCCH members have counterparts in higher plants, supporting the ancient origin of the CCCH family and suggesting subfamily IX might be a derived subfamily specific to advanced plants.
Identification and Analysis of the Rice CCCH Gene Family
Rice, a crucial crop and model monocot, also possesses a large CCCH gene family. We employed similar bioinformatics approaches to identify and characterize CCCH genes in rice. A Perl program and BLAST searches identified 67 CCCH genes in the rice genome. Phylogenetic analysis of rice CCCH proteins revealed 8 subfamilies with high bootstrap support, exhibiting similar gene structures and zinc finger motifs within each subfamily. Two large subfamilies in rice mirrored corresponding subfamilies in Arabidopsis.
Unlike Arabidopsis, the chromosomal distribution of CCCH genes in rice is skewed towards chromosomes 1-7. Phylogenetic analysis identified 13 pairs of putative duplicated genes in rice, representing 39% of the family, suggesting genome duplication events also played a role in rice CCCH family expansion.
Comparative Phylogenomics Reveals Ancient Origins and Orthologous Relationships
To examine the evolutionary relationships between CCCH families in Arabidopsis and rice, we performed a combined phylogenetic analysis. This analysis revealed 20 subfamilies, with 8 subfamilies containing members from both Arabidopsis and rice, indicating an ancient origin of these subfamilies predating the monocot-eudicot divergence. We identified 30 putative orthologous gene pairs between Arabidopsis and rice. Paralogous relationships observed in individual species were largely maintained in the combined tree.
Despite differences in genome size and gene number between Arabidopsis and rice, the CCCH gene family size is remarkably similar (68 and 67 genes, respectively). The CCCH family is one of the largest in plants, indicating its functional importance. The C-X7–8-C-X5-C-X3-H motif is the most abundant in both species, suggesting it might be an ancestral CCCH motif. We also identified five novel CCCH motif variations, expanding the known diversity of this family.
Figure 5: Arabidopsis CCCH protein structures. This schematic illustrates the domain organization of all identified Arabidopsis CCCH proteins, depicting the number and types of CCCH zinc fingers, and providing a visual representation of the finger family’s structural diversity.
Sequence alignment of 302 CCCH motifs from Arabidopsis and rice revealed complete conservation of the four zinc-coordinating residues (three cysteines and one histidine), as well as enrichment for glycine and phenylalanine. These conserved features allowed us to refine the definition of a CCCH motif as C-X4–15-C-X4–6-C-X3-H, characterized by glycine-rich and phenylalanine-rich regions.
Figure 6: CCCH zinc finger motif sequence logo. This sequence logo visually represents the conserved amino acids within CCCH motifs from Arabidopsis and rice, highlighting the prevalence of cysteine, histidine, glycine, and phenylalanine in the finger family motif.
Tissue-Specific Expression Patterns Suggest Diverse Functions
Gene expression patterns can provide insights into gene function. We analyzed the expression of Arabidopsis and rice CCCH genes across different tissues (root, leaf, flower, seed) using MPSS and EST data. Most CCCH genes exhibit broad expression patterns, with 33 Arabidopsis and 36 rice genes expressed in all tissues examined. However, a subset of genes shows tissue-specific expression.
Figure 7: Expression patterns of Arabidopsis and rice CCCH genes. This heatmap visualizes the expression profiles of CCCH genes in different tissues (root, inflorescence, leaf, seed) in Arabidopsis and rice, illustrating the diverse expression patterns within the finger family across plant tissues.
In Arabidopsis, AtC3H50 and AtC3H68 are root-specific, while others show preferential expression in inflorescences (AtC3H18, AtC3H35, AtC3H43, OsC3H36, OsC3H39), leaves (AtC3H45, OsC3H28, OsC3H30), or seeds (AtC3H2, AtC3H5, AtC3H13, AtC3H15, AtC3H54, OsC3H1, OsC3H52). Duplicated gene pairs often exhibit divergent expression patterns, supporting functional diversification following gene duplication. The diverse expression patterns of CCCH genes suggest their involvement in a wide range of biological processes across different plant tissues and developmental stages.
Subfamily IX and I: Stress-Responsive CCCH Zinc Finger Proteins
Subfamily IX in Arabidopsis and subfamily I in rice are the largest CCCH subfamilies in their respective genomes. Members of these subfamilies typically contain two CCCH motifs, often C-X7–8-C-X5-C-X3-H and C-X5-C-X4-C-X3-H in tandem. A conserved C-X5-C-X4-C-X3-H motif and a putative CHCH motif are characteristic of these proteins. Intriguingly, genes in these subfamilies lack introns.
Phylogenetic analysis further divides Arabidopsis subfamily IX into two subgroups. Subgroup 2 members contain ankyrin (ANK) repeat motifs, known for mediating protein-protein interactions. Five out of six Arabidopsis genes encoding both ANK repeats and zinc finger domains belong to CCCH subfamily IX, highlighting the unique domain architecture of this subfamily. Combined phylogenetic analysis of Arabidopsis subfamily IX and rice subfamily I revealed three subgroups, with limited orthologous relationships between species, suggesting potential species-specific evolution within these subfamilies.
Figure 8: Subfamily IX motif analysis. This figure details the sequence characteristics of subfamily IX, showing alignments of CCCH motifs, ankyrin repeats, and putative NES sequences, revealing the domain features within this finger family subgroup.
Database searches predict nuclear localization for all members of subfamily IX and I. Putative Nuclear Export Signals (NES) were identified in all subfamily IX members, suggesting they may function as nucleocytoplasmic shuttle proteins involved in signal transduction. Microarray and RT-PCR data revealed that subfamily IX genes in Arabidopsis are responsive to various abiotic stresses (salt, cold, mannitol, ABA, hypoxia, osmotic stress), indicating their roles in stress responses.
Figure 9: Phylogenetic trees of subfamilies IX and I. These phylogenetic trees illustrate the relationships within Arabidopsis subfamily IX and between Arabidopsis subfamily IX and rice subfamily I, revealing subgroups and evolutionary divergence within this stress-responsive finger family clade.
Figure 10: Stress-responsive expression of subfamily IX genes. This figure shows microarray and RT-PCR data illustrating the expression patterns of subfamily IX genes in Arabidopsis under various abiotic stresses and ABA treatment, demonstrating the finger family’s role in stress response.
CCCH Proteins as RNA-Binding Factors: Structure and Potential Targets
The CCCH domain is known to mediate RNA binding. Sequence alignment revealed that AtC3H14, AtC3H15, OsC3H9, and OsC3H39 share high sequence similarity with mammalian TIS11D, a well-characterized RNA-binding protein. These plant proteins also contain two tandem C-X8-C-X5-C-X3-H CCCH zinc fingers, similar to TIS11D. Structural modeling of the AtC3H14 CCCH domain based on the TIS11D-RNA complex suggests a conserved RNA-binding mode. Key residues involved in RNA binding in TIS11D, such as the KTEL(V) motif and aromatic residues, are also conserved in these plant CCCH proteins, suggesting a similar RNA-binding mechanism.
Figure 11: RNA-binding domain analysis of AtC3H14. This figure includes sequence alignments highlighting conserved residues with TIS11D and a structural model of the AtC3H14 CCCH domain in complex with RNA, illustrating the potential RNA-binding mechanism of this finger family member.
In mammals, CCCH proteins like TTP and TIS11D regulate mRNA stability by binding to AU-rich elements (AREs) in the 3′-UTR of target mRNAs. We identified 200 Arabidopsis genes containing AREs in their 3′-UTRs, suggesting potential targets for CCCH-mediated RNA regulation in plants. Further experiments are needed to confirm the RNA-binding activity of plant CCCH proteins and identify their specific mRNA targets.
Conclusion
This comprehensive study provides a detailed characterization of the CCCH zinc finger protein family in Arabidopsis and rice. We identified 68 CCCH genes in Arabidopsis and 67 in rice, classified them into subfamilies based on phylogenetic relationships, and analyzed their conserved motifs, gene structures, and expression patterns. Our findings highlight the evolutionary conservation of the CCCH family, the role of gene duplication in its expansion, and the diverse expression patterns suggesting a wide range of functions. Subfamily IX in Arabidopsis and subfamily I in rice emerged as stress-responsive subfamilies with potential roles in signal transduction, and structural modeling suggests that plant CCCH proteins are likely to function as RNA-binding proteins, potentially regulating gene expression at the post-transcriptional level. This study lays the groundwork for future functional studies to dissect the specific roles of individual CCCH proteins in plant growth, development, and stress responses, further illuminating the importance of this large and diverse finger family in plant biology.
