Flanking Sequence Cotranscriptionally Regulates Twister Ribozyme Activity.

Area of Science:

• Molecular Biology
• Biochemistry
• RNA Biology

Background:

• Ribozymes are RNA molecules with catalytic activity, but their regulatory mechanisms are less understood than protein enzymes.
• Twister ribozymes, a class of nucleolytic RNAs, have known catalytic properties, but their regulation within native sequences is understudied.
• Naturally occurring ribozymes are not isolated and their activity can be influenced by adjacent genomic sequences.

Purpose of the Study:

• To investigate the regulatory role of flanking sequences on twister ribozyme activity in rice (Oryza sativa).
• To identify specific twister ribozymes whose activity is modulated by their native genomic context.
• To explore potential mechanisms of ribozyme regulation and external control applicable to synthetic biology.

Main Methods:

• Analysis of published RNA-sequencing data sets from rice.
• Structure probing techniques to investigate RNA conformation.
• Cotranscriptional cleavage assays to measure ribozyme activity.
• Oligonucleotide-mediated rescue experiments.

Main Results:

• Differential regulation of twister ribozymes by flanking sequences was observed.
• The Osa 1-2 ribozyme showed no interaction with flanking sequences.
• Flanking sequences of Osa 1-3 and Osa 1-8 ribozymes formed inactive ribozymogen conformations, attenuating self-cleavage.
• Antisense oligonucleotide addition rescued the activity of the Osa 1-3 ribozymogen.

Conclusions:

• Flanking sequences play a significant role in differentially regulating twister ribozyme activity in vivo.
• Ribozymogen formation represents a plausible mechanism for natural ribozyme regulation.
• External control of ribozymogens, demonstrated with antisense oligonucleotides, has implications for synthetic biology and gene regulation.