Large-scale mutational analysis uncovers molecular mechanisms governing dual RNA functions in transposons

Transposons are among the most abundant and diverse mobile genetic elements in nature. A unique class termed IStrons encodes a transposase for transposon mobility, an RNA-guided nuclease for maintenance, and a self-splicing group I intron for element removal from host mRNA. However, it is unclear how a single polynucleotide sequence balances these distinct biochemical functions. Here we employed pooled library mutagenesis coupled with high-throughput sequencing to systematically dissect the molecular determinants of IStron transposition, RNA-guided DNA cleavage, and self-splicing. We found that the terminal trinucleotide of the transposon right end is constrained by all three functions, identifying a molecular convergence point. Cross-assay comparisons revealed that most variants maintained or lost activity across multiple assays simultaneously. However, a subset selectively retained one activity while losing another, revealing antagonism between DNA cleavage and splicing governed by guide RNA structural stability. Increased GC content at the base of the guide RNA 3 terminal stem-loop abolished splicing while maintaining DNA cleavage, and the properly folded guide RNA sterically occluded alternative splice sites, ensuring splicing accuracy across variable flanking contexts. Thus, IStron transcripts overcome an inherent trade-off between guide RNA maturation and splicing, with RNA structural stability as the primary determinant of pathway choice.