Structure–Function Relationships of Cid1 Poly(U) Polymerase: Catalysis, Substrate Recognition, Engineering, and Applications

RNA 3′-end modification is an important regulatory mechanism that influences RNA processing, stability, localization, function, and degradation. Beyond its biological roles, RNA 3′-end modification has become a valuable tool in biotechnology, enabling applications in RNA labelling, sequencing, engineering and oligonucleotide synthesis. Terminal nucleotidyltransferases are key enzymes responsible for template-independent nucleotide addition to RNA molecules. Among these enzymes, Schizosaccharomyces pombe Cid1, a poly(U) polymerase, has emerged as an important model for understanding RNA-specific terminal nucleotidyltransferase activity. Structural and biochemical studies have revealed how nucleotide recognition, metal-ion-dependent catalysis, RNA positioning, and conformational dynamics regulate Cid1-mediated uridylation. Recent advances in protein engineering, molecular simulation, machine learning, and generative protein design have further expanded Cid1 functionality, generating variants with improved activity, altered substrate specificity, and potential applications in enzymatic RNA synthesis. This review summarizes the biological functions, structural characteristics, catalytic mechanisms, protein engineering strategies, and emerging biotechnological applications of Cid1, as well as the remaining challenges, highlighting its potential as a programmable platform for RNA 3′-end functionalization and enzymatic RNA oligonucleotide synthesis.