Integrated Comparative Analysis Reveals Structural Diversification and Functional Associations of Arabidopsis DEAD-Box RNA Helicases

DEAD-box RNA helicases (RHs) constitute one of the largest and most conserved families of RNA metabolism factors in eukaryotes, yet the evolutionary relationships and functions of many plant members remain unresolved. We present an integrated comparative analysis of the Arabidopsis thaliana (Arabidopsis) DEAD-box RH family by combining structural curation, orthology inference, comparative analyses of protein features, and cross-species comparisons of biological pathway enrichment among co-expressed genes. Structural curation established a reference set of 53 intact genes and four putative pseudogenes (RH43, RH44, RH45, and RH55) for subsequent analyses. Orthology inference using the DRSC Integrative Ortholog Prediction Tool (DIOPT) identified high- and moderate-confidence orthologous relationships, revealed conserved helicase lineages and putative lineage-specific expansions, and enabled systematic comparisons with Saccharomyces cerevisiae (yeast) and Homo sapiens (humans). Comparative analyses of protein features revealed that non-canonical DExD-box motif variants occur in 10 of the 57 Arabidopsis, three of the 26 yeast, and five of the 38 human DEAD-box RHs. Although the DECD variant appears to be shared across plant, fungal, and metazoan lineages, other variants show restricted or patchy distributions, as exemplified by the DEVD variant, whose distribution is consistent with its independent emergence in several lineages, indicating that the canonical DEAD sequence is not universally conserved. Annotated accessory motifs and domains were identified in only a small subset of proteins, although annotation-based counts probably underestimate their frequency, and the helicase core architecture was broadly conserved. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of genes co-expressed with Arabidopsis DEAD-box RHs and their orthologs provided a basis for generating functional hypotheses for previously uncharacterized proteins, even in the absence of prior experimental evidence of ortholog function. Although these hypotheses require further investigation and experimental validation, the findings provide an integrated framework for investigating the structural and functional diversification of the Arabidopsis DEAD-box RH family. This approach may also be extended to other poorly characterized multigene families.