Engineering Site-Specific Fluorescently-Labeled IEPs to Monitor Group II Intron RNP Assembly

TL;DR

Researchers developed a method to fluorescently label proteins and RNA in group II introns to study how they assemble into functional complexes in real time.

Contribution

A novel site-specific fluorescent labeling method for IEPs and RNA in group II introns that preserves their biological activity.

Findings

Cysteine-to-methionine mutations in IEP enabled site-specific fluorescent labeling without affecting splicing or retromobility.

Fluorescent labeling of IEP and RNA allowed real-time monitoring of RNP assembly in native conditions.

The labeled system revealed structural flexibility in IEP-intron interactions during splicing and retromobility.

Abstract

Group II introns are self-splicing ribozymes that excise themselves from precursor RNA and mobilize to new DNA loci via retromobility. Central to this mechanism is the intron-encoded protein (IEP), a multidomain maturase and reverse transcriptase that facilitates splicing and formation of a ribonucleoprotein (RNP) complex. As an RNP, the intron can be integrated into a DNA target by two reverse splicing steps followed by IEP- catalyzed complementary DNA (cDNA) synthesis. Group II introns progress through various conformational states as they undergo splicing and retromobility, therefore, we set out to develop tools to monitor RNP assembly. We engineered a suite of cysteine-to-methionine mutations in the IEP of the group IIC intron Ta.it.I1, which requires the IEP for lariat splicing and forming RNPs. These mutations enabled site-specific fluorescent labeling using maleimide-thiol chemistry without compromising splicing or retromobility activity. We also labeled the intron RNA through addition of FAM- labeled UTPs during in vitro transcription. Labeled IEPs retained maturase and reverse transcriptase functionality.

The fluorescently labeled IEP and RNA system were successfully used to monitor IEP-intron binding for RNP assembly in native conditions. These findings demonstrate the structural flexibility of IEP-intron interactions during splicing and retromobility, establishing a powerful tool for real- time visualization of RNP assembly.