HDV Family of Self-Cleaving Ribozymes - Progress in Molecular Biology and Translational Science - page 142

unique families from the genomes of the mosquito Anopheles gambiae and the
purple sea urchin Stronglyocentrotus purpuratus . Active motifs were also iden-
tified in the lamprey eel Petromyzon marinus , the lancelet Branchiostoma
floridae , and two nematodes Caenorhabditis japonica and Pristionchus pacificus .
Outside of eukaryotes, additional HDV-like ribozymes were found in the
human gut commensal bacterium Faecalibacterium prausnitzii , and the insect
Chilo Iridescent Virus type 6 (CIV). 3 These newly defined HDV-like motifs
subsequently served as starting points for additional sequence and structure-
based searches for HDV ribozymes.
5.8. The A. gambiae ribozymes
A number of self-cleaving sequences have been detected in the African
malaria mosquito A. gambiae . When fitted to an HDV secondary structure
model, the ribozymes can be differentiated into two classes, drz-Agam-1 and
drz-Agam-2 ( Fig. 4.7 ). Isolates of the drz-Agam-1 family are approximately
the same length and structure as the HDV ribozymes, whereas the drz-
Agam-2 family is farther removed from the canonical HDV secondary
structure.
The J4/2 region of the drz-Agam-1 family of ribozymes is of particular
interest due to the presence of a second cytosine adjacent to the catalytic
cytosine. Self-scission is eliminated upon mutation of both of these residues
to uridines; however, C to U substitution of just the upstream cytosine
yields a construct that exhibits only residual activity (C.T. Webb, personal
communication). This suggests flexibility in the HDV core, where
both cytosines are potentially positioned to act in catalysis. However, the
detailed mechanism of self-scission for this variant ribozyme has not been
studied.
The drz-Agam-2 family differs from the drz-Agam-1 sequences in that it
possesses a much larger P4/L4 domain and a large insertion in the J1/2
region of the secondary structure. The J1/2 regions of the genomic and anti-
genomic HDV ribozymes also vary in length, although not to the extent
observed between the mosquito families. Among the drz-Agam-2
sequences, the J1/2 peripheral domain is approximately 50 nt in length, rep-
resenting approximately one-third of the total sequence space for the struc-
ture. Computational modeling predicts that the J1/2 insertion forms
extensive base-pairing interactions leading to the creation of a sixth helix
in the secondary structure. The formation of the sixth helix likely enhances