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

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.

1

Group IIC intron-encoded protein sequence alignment showing conserved domains. (A) α-fold3 representation of Ta.it.I1 IEP colored by canonical RT regions of fingers (gray), palm (light pink), and the X/DBD regions of the thumb (green). The catalytic YADD resides within the palm region of the RT domain, colored in cyan, and the cysteines are colored in purple. The box below the structure represents the sequence of the RT and the location of the cysteine residues (purple). (B) Sequences of four group IIC intron-encoded protein sequences (Ta.it. RT, O.i. RT, MarathonRT, GsI RT) showing conservation between the RT domain (light pink arrow) and X and DBD domains (green arrow). More yellow indicates less conservation while more blue indicates more conservation. The catalytic YADD is highlighted in cyan and all endogenous cysteine residues for each RT are highlighted in orange with their names in purple.

2

Purification and in vitro splicing activity of cysteine-mutated IEPs. (A) Cartoon depiction of the mutant IEPs and the sites of cysteine mutagenesis with their respective names below. (B) SDS-PAGE of purified IEP cysteine mutants, WT, and YAAA. (C) Schematic of the two competing splicing pathways of Ta.it.I1 intron with and without IEP. (D) 4% PAGE analysis of in vitro splicing activity of Ta.it.I1 intron RNA with IEP cysteine mutants, YAAA mutant, and WT under standard splicing conditions. The bar graph (right) compares the average percent lariat formed with each IEP cysteine mutant and YAAA with WT IEP. The mean was calculated with five replicates, and the error bars represent the standard deviation (SD). The numbers above the bar graph correspond with their respective gel lane. Uncropped versions of (B, D) is in Figures S1 and S2, respectively.

3

In vitro reverse transcription activity of cysteine-mutated IEPs. (A) Schematic of the reverse transcription assay. (B) Bar graph of average relative fluorescence measured in each cysteine-mutated IEP, WT, and YAAA. The average fluorescence was calculated with five replicates, and the error bars represent the SD.

4

In vitro retromobility of cysteine-mutated IEPs. (A) Schematic of the retromobility assay. The dashed lines represent a close-up visualization of limited cDNA synthesis with addition of dATPs. (B) Cysteine mutants, YAAA, and WT RNP were tested for integration into the fluorescently labeled DNA substrate with and without dATP. Green bands indicate FAM detection of the 3′ exon only. Yellow bands indicate detection of both Cy5 and FAM, showing that the 5′ and 3′ exons of the DNA substrate are detected together. (C) A bar graph (left) compares the average percent of 3′ lariat-DNA intermediate produced (green band only) of IEP cysteine mutants and YAAA with WT. Another bar graph (right) compares the average complete integration product with limited cDNA synthesis by dATP addition of IEP cysteine mutants and YAAA with WT (yellow band). The averages were calculated with five replicates, and the error bars represent the SD. The numbers above the columns of each bar graph correspond to the band lane in the above retromobility gel in (B). (D) A time course analysis of full-length cDNA synthesis was performed comparing C345M and C214,220M with WT after addition of dNTP. The band colors follow the same as in (B), however the smearing of the yellow indicates different lengths of synthesized cDNA. The bar graph (right) shows the average full-length product at each time point for C345M and C214,220M relative to WT. Averages were calculated from three replicates, and error bars represent the standard deviation (SD). Uncropped versions of panels (B, D) are shown in Figures S4 and S5, respectively.

5

Functional maleimide labeling of cysteine-mutated IEPs (A) Cartoon depiction of representative cysteine-mutated IEP’s labeled with AF647 compared to WT IEP. (B) SDS-PAGE comparing cysteine mutant IEPs samples before and after fluorescently labeling with a functional maleimide containing AF647. Coomassie stains (top gel) show the protein population in each sample, with the main purified component at 68.6 kDa. Cy5 scan (bottom gel) is the same gel but scanned for detection of IEP labeled with AF647, showing the same purified labeled band at 68.6 kDa. (C) 4% Denaturing PAGE analysis of in vitro splicing activity of Ta.it.I1 intron RNA with labeled IEP cysteine mutants compared to unlabeled and WT under standard splicing conditions. The bar graph (right) compares the average lariat produced between labeled and unlabeled IEP, which is also compared back to WT. The average lariat value was calculated from five replicates, and error bars represent the standard deviation (SD). The numbers above the bar graph correspond to the respective gel lanes. Uncropped versions of panels (B, C) are shown in Figures S6 and S7, respectively.

6

Functional AF647 IEP and FAM body-labeled intron RNA. (A) Cartoon representation of the fluorescent-labeled RNA method. FAM-labeled UTPs are supplemented to the in vitro transcription reaction to yield FAM body-labeled intron RNA. (B) 4% Denaturing PAGE analysis of in vitro splicing activity with FAM-labeled RNA with a titration of either AF647-labeled C220,345M or E271C IEP. The top gel is stained with ethidium bromide to show the RNA as it is spliced, and the bottom gel is a Cy2 scan to excite the FAM fluorophore, showing the retention of fluorescence after lariat splicing. (C) 5% Native PAGE analysis of in vitro splicing activity with FAM-labeled RNA comparing the RNP formation dynamics under low and high molar excess of AF647 C220,345M and E271C IEP. Left gel shows the Cy5 scan of the AF647 IEP present in the gel, middle gel shows the Cy2 scan of the FAM RNA present in the gel, and the right gel shows the composite image of the Cy2 and Cy5 scans showing how the bands overlap and interact. Uncropped versions of (B, C) is in Figures S8 and S9, respectively.