HDV Family of Self-Cleaving Ribozymes - Progress in Molecular Biology and Translational Science - page 152

length of the helices for the various families and the composition of the J4/2
region. A slightly longer P2 helix of 8 bp, a short, 5 bp P4 helix, no J1.1/4
region, and a J4/2 active site of CGAA composition characterize the drz-
Spur-1 family. In the drz-Spur-2, -3, and -4 families of sequences, the P2
helix is 7 bp in length. Drz-Spur-2 is unique in that it also possesses a
J1.1/4 segment and a J4/2 region of CUAA. The only other family of
S . purpuratus ribozymes with a J1.1/4 region is the drz-Spur-3 family, which
is the only known HDV-like ribozyme with a 6 bp P1 helix. The J4/2
region of this family is composed of CCAA. Much like in the drz-Agam-1
family of sequences, the second cytosine in this region also appears to func-
tion in catalysis, as self-cleavage activity is lost only if both cytosines are
mutated. Finally, the drz-Spur-4 family is distinguished by its extended
P4 helix and “CUGA” J4/2 region. In all of the drz-Spur families, multiple
copies of the sequences are present throughout the sea urchin genome.
By far, the most common class of sea urchin ribozymes is the drz-Spur-1
family. BLAT and BLAST analysis of the drz-Spur-1 sequence identifies
well over 30 full-length copies dispersed throughout the genome. One
copy, which is transcribed in vivo (ESTCD324081), is found in the first exon
of a predicted non-LTR retrotransposase gene. Given the recently identified
role of HDV-like ribozymes in the cotranscriptional processing of
retrotransposons, it is likely that the drz-Spur-1 ribozyme plays a crucial part
in the transposition event. 3 Association with such an element might also
explain the much higher copy number of drz-Spur-1 ribozymes relative
to the other drz-Spur families.
A number of the putative drz-Spur-1 ribozymes contain mutations that
would render them inactive. These include active-site cytosine to uridine
mutations, deletions on the 3 0 side of the P1 helix, and mutations to the gua-
nosine nucleotide that forms the 3 0 side of the cleavage site. However, the
latter mutation may not prove fatal to self-cleavage activity, as the drz-
Spur-1 sequences contain a second guanosine adjacent to the cleavage site
guanosine that could serve to shift the scission location one nucleotide
downstream. This would yield a 6 nt P1 helix in the drz-Spur-3 family of
sequences, which show robust self-cleavage despite the shortened P1 region
( k obs ¼ 12 h 1 in 1 mM Mg 2 þ at 22 C).

Unlike many of the other HDV-like ribozymes identified through second-
ary structure searches, the ribozymes from S . purpuratus frequently appear within
or near genes or predicted genes. Three copies of drz-Spur-1 appear in the 22nd
intron of a predicted homeobox gene, although one of these copies contains an
active-site C53Umutation. Drz-Spur-2 and drz-Spur-4 both appear four times