Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (3 results)

  • Thesis
  • Peer Reviewed
UC Irvine Electronic Theses and Dissertations (2014)

Investigating the influence of peripheral domains on the kinetic rates of HDV-like ribozyme self-cleavage is important to discovering their biological relevance. The following work investigates the effects of peripheral elements on the self-scission of two HDV-like ribozymes, the mouse CPEB3 ribozyme and the mosquito drz-Agam-2-1.

The CPEB3 ribozyme, located in an intron of the mammalian CPEB3 gene, is the first HDV-like ribozyme discovered in a genome other than the virus. ESTs from human cells suggest ribozyme activity in vivo and demonstrate tissue-specific expression1; the level of activity in human brain tissue suggests another factor, such as a chaperone, increases ribozyme activity by minimizing misfolding2. The rates of the co-transcriptional self-scission of the CPEB3 ribozyme were determined using sequences varying by the number of nucleotides surrounding the ribozyme. Experimental rates range from 0.13 ± 0.029 to 0.46 ± 0.020 min-1 with the order of the constructs being -233/72, -197/72, -197/72/21, -233/72, -197/72/165, - 233/72/165, -10/72, -49/72/165 and -257/72/328. The findings here demonstrate that CPEB3 ribozymes are kinetically fast-reacting and suggest a relationship between flanking sequences and rate.

The drz-Agam-2-1 ribozyme was the first naturally occurring ribozyme to contain a helix in the J1/2 domain3, a domain shown to indirectly effect ribozyme self-cleaving4. The rate of self-cleavage increased with step-wise reintroduction of the helix into drz-Agam-2-1. Experiments correlated the stability derived from J1/2 to catalysis and provide an explanation for the ribozyme's Hill coefficient of n=1. The data shows the J1/2 structured element to impact ribozyme catalysis through stabilization of the catalytic core.

    Cover page: The influence of peripheral elements on the kinetics of HDV-like ribozymes
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2016)
    Self-cleaving ribozymes fold into intricate structures, which orient active site groups into catalytically competent conformations. Most ribozyme families have distinct catalytic cores stabilized by tertiary interactions between domains peripheral to those cores. We show that large hepatitis delta virus (HDV)-like ribozymes are activated by peripheral domains that bring two helical segments, P1 and P2, into proximity - a "pinch" that results in rate acceleration by almost three orders of magnitude. Kinetic analysis of ribozymes with systematically altered length and stability of the peripheral domain revealed that about one third of its free energy of formation is used to lower an activation energy barrier, likely related to a rate-limiting conformational change leading to the pre-catalytic state. These findings provide a quantitative view of enzyme regulation by peripheral domains and may shed light on the energetics of allosteric regulation.
    • 1 supplemental file
    Cover page: Topological constraints of structural elements in regulation of catalytic activity in HDV-like self-cleaving ribozymes
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2024)
    A self-cleaving ribozyme that maps to an intron of the cytoplasmic polyadenylation element-binding protein 3 (Cpeb3) gene is thought to play a role in human episodic memory, but the underlying mechanisms mediating this effect are not known. We tested the activity of the murine sequence and found that the ribozymes self-scission half-life matches the time it takes an RNA polymerase to reach the immediate downstream exon, suggesting that the ribozyme-dependent intron cleavage is tuned to co-transcriptional splicing of the Cpeb3 mRNA. Our studies also reveal that the murine ribozyme modulates maturation of its harboring mRNA in both cultured cortical neurons and the hippocampus: inhibition of the ribozyme using an antisense oligonucleotide leads to increased CPEB3 protein expression, which enhances polyadenylation and translation of localized plasticity-related target mRNAs, and subsequently strengthens hippocampal-dependent long-term memory. These findings reveal a previously unknown role for self-cleaving ribozyme activity in regulating experience-induced co-transcriptional and local translational processes required for learning and memory.
      Cover page: Inhibition of Cpeb3 ribozyme elevates CPEB3 protein expression and polyadenylation of its target mRNAs and enhances object location memory.
      Top