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

## TL;DR

This study shows that replacing cysteine residues in a protein encoded by a group II intron does not hinder its splicing or mobility functions and allows for site-specific labeling.

## Contribution

The study demonstrates that cysteine residues in the IEP can be substituted without impairing function and enables site-specific fluorescent labeling.

## Key findings

- Cysteine-to-methionine mutations in the IEP retained near wild-type splicing efficiency.
- A thumb domain mutation increased reverse transcription activity, while YADD motif substitutions reduced it.
- Labeled IEPs maintained activity and enabled real-time monitoring of RNA binding and RNP assembly.

## Abstract

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 Ta.it.I1 from
the thermophile Thermoanaerobacter italicus 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.