Cysteine Mutagenesis of a Group II Intron-Encoded Protein Supports Splicing, Mobility, and Site-Specific Labeling.

Group II introns are self-splicing ribozymes that excise themselves from precursor RNA and integrate into new DNA locations through retromobility. Splicing is facilitated by an intron-encoded protein (IEP), a multidomain reverse transcriptase that enhances ribozyme activity and promotes formation of lariat intron-IEP ribonucleoprotein (RNP) complexes. In this study, we examined the role of conserved cysteine residues in the IEP of the group IIC intron I1 from the thermophile by generating cysteine-to-methionine mutants. All variants retained near wild-type splicing efficiency, indicating that cysteine substitution does not impair maturase function. A mutation in the thumb domain significantly enhanced reverse transcription (RT) activity, whereas substitutions flanking the YADD catalytic motif led to reduced activity. Despite these variable RT effects, all mutants retained the ability to complete both steps of forward intron self-splicing and subsequently perform reverse splicing into DNA targets. Complete removal of native cysteines enabled site-specific fluorescent labeling of the IEP using maleimide-thiol chemistry without disrupting splicing or retromobility. Labeled IEPs retained activity and were successfully used to monitor RNA binding and RNP assembly under native conditions. These findings highlight the structural flexibility of IEP-intron interactions and demonstrate that site-specific IEP labeling enables real-time visualization of RNP assembly and dynamics.