HDV Family of Self-Cleaving Ribozymes - Progress in Molecular Biology and Translational Science - page 137

to cleave approximately 250-fold faster simply by mutating a single cytosine
residue located 2 from the cleavage site to an A. 98 This mutation disrupts the

formation of an alternate P1/P1.1 helix that extends the 3 0 side of P1 helix
through the P1.1 region, disrupting the nested double pseudoknot essential
for activity. Although the roles that flanking regions play in ribozyme folding
have currently been investigated only in the primate CPEB3 motif, it is likely
that similar upstream or downstream modifiers exist in other mammalian
sequences that either speed up or slow down the cleavage reaction.

5.4. Expression and biological roles of the CPEB3 ribozyme
The cytoplasmic polyadenylation element binding proteins are important for
translation regulation, and CPEB3 is specifically responsible for binding a
novel translation-regulating signal that consists of a stem-loop with a U-rich
bulge. 99,100 Expression of CPEB3 occurs in a wide range of tissues and is
increased in mouse hippocampus following kainate-induced seizure. 101 The
CPEB3 protein has been shown to repress translation of mRNAs containing
its binding motif, perhaps because it interacts with features other than the con-
served structures and proteins essential for CPEB -induced polyadenylation
and thus prevents the mRNA from being polyadenylated and translated. 100
The CPEB3 ribozyme is located between the first and second coding
exons of the CPEB3 gene. This places it in the second intron of the gene
because the 5 0 UTR of the CPEB3 mRNA is also spliced. Depending on
the species, the ribozyme motif appears 10-25 kb upstream of the second
coding exon. An RNA polymerase molecule moving at 2000-4000 nt/min
would take 3-13 min to reach the second exon. 102,103 Although the initially
reported in vitro cleavage rate (1 h 1 in 2 mM Mg 2 þ at 37 C) indicates that
the ribozyme-catalyzed self-scission reaction would not interfere with
cotranscriptional splicing, recent data suggest that the in vivo reaction is
much faster and could affect pre-mRNA stability before tagging or splicing
of the second exon. 2,98 Indeed, a number of expressed sequence tags (ESTs)
have been identified from Mus musculus (DT917655.1, DT914013.1,
BY750066.1) and opossum (EC326704.1) that contain 5 0 termini identical
to the in vitro cleavage site of the ribozyme, providing strong evidence that
the ribozyme is expressed and self-cleaves in vivo . The fact that these tran-
scripts can be detected at all suggests that the ribozyme stabilizes the 5 0 end of
the transcript, protecting it from 5 0 to 3 0 exonucleases following the cleavage
event. Furthermore, rapid amplification of cDNA ends (5 0 RACE) exper-
iments identified self-cleaved ribozymes in human testis and placenta, as well