HDV Family of Self-Cleaving Ribozymes - Progress in Molecular Biology and Translational Science - page 147

and the 2 0 OH of the conserved A78 residue in J4/2. 34-36 In vitro s election
experiments have shown that variants that maintain base pairing in the P3 are
tolerated, although wild-type sequences appear with much higher fre-
quency. 44 Formation of a properly base-paired P3 is essential for catalysis;
therefore, the observed conservation may instead reflect the rarity of two
compensatory mutations occurring in this region in a single replicative
event. 41
Unlike the P3 helix, the P2 helix varies greatly in both nucleotide com-
position and length among the HDV-like motifs. In the R2 ribozymes, this
region is potentially longer than the six to eight base-pair norm, suggesting,
in conjunction with its high level of conservation, that it has an important
role in catalysis or retrotransposition. Comparable conservation for this
motif is not found among isolates from the drz-Agam-2 family. 3 The high
levels of in vivo expression of the Drosophila R2 ribozymes provide ample
opportunity for random mutations to arise, but that none of these mutations
has accumulated in the P2 helix suggests an additional role for this motif.
The observed conservation of P2 in the R2 ribozyme may be linked to a
role in translation. Since mRNA generated cotranscriptionally from a larger
transcript via the actions of a self-cleaving ribozyme would not possess the
5 0 methyl-guanosine cap that ensures stability of the message and promotes
translation, other methods must be employed to achieve protection and
begin translation. Resistance to degradation is represented at least in part
by the unusual high stability of the HDV ribozyme structure, as folding
and self-cleavage are observed even in high concentrations of denaturants
for the viral sequences and CPEB3 . 2,52,127 Translation initiation, however,
can only be reconciled if the ribozyme is acting as an internal ribosome entry
site (IRES). There exist several classes of IRESes with the underlying fea-
tures that they form complex secondary structures, contain a recognizable
codon, and are capable of starting translation in the absence of some or all
of the canonical translation initiation factors. 128
Recent evidence suggests that the retrotransposon ribozymes are indeed
capable of acting as IRESes. 126 Constructs containing either wild-type or
inactive C/U mutant ribozymes upstream of a luciferase reporter gene
Figure 4.8—Cont'd to produce the R2 protein, which nicks the target DNA and yields a
free, 3' OH. The free 3' OH is used in (4) target-primed reverse transcription to create the
R2 cDNA, which forms the template for (5) second-strand synthesis of the R2 DNA. The
full-length R2 retrotransposon is then (6) reinserted into the genome to yield an active
retrotransposon at a new 28S rDNA locus.