Two group I ribozymes with different functions in a nuclear rDNA intron

Area of Science:

• Molecular Biology
• RNA Catalysis
• Intron Splicing

Background:

• The DiSSU1 mobile intron in Didymium iridis contains two catalytic RNA elements.
• These elements were previously identified but their specific functions were not fully elucidated.

Purpose of the Study:

• To characterize the catalytic functions of the two RNA elements within the DiSSU1 intron.
• To determine the roles of GIR1 and GIR2 in intron processing and potential gene expression.

Main Methods:

• In vitro transcription and catalytic assays of RNA elements.
• Site-directed mutagenesis of specific secondary structure elements (P7 pairing segment, P2 stem/loop).
• Analysis of mutation effects on ribozyme activity (self-splicing and hydrolysis).

Main Results:

• Both GIR1 and GIR2 were confirmed as group I ribozymes with distinct catalytic activities.
• GIR2 performs the self-splicing reactions of the intron.
• GIR1 catalyzes hydrolysis at an internal processing site (IPS-1), representing the smallest known natural group I ribozyme with this function.
• Mutations in the P7 segment abolished the respective activities of GIR2 (self-splicing) and GIR1 (hydrolysis).
• Deletions in the P2 stem/loop did not affect GIR2 self-splicing, supporting a model where P2 brings the 5' splice site to the catalytic core.

Conclusions:

• GIR1 and GIR2 are functionally differentiated group I ribozymes within the DiSSU1 intron.
• GIR1's novel hydrolytic function is hypothesized to be involved in generating mRNA for the intron-encoded endonuclease I-DirI.
• The findings provide insights into the diverse roles and mechanisms of mobile introns and ribozymes.