The Role of snRNAs in Spliceosomal Catalysis - Progress in Molecular Biology and Translational Science - page 196

in fact dispensable for the first step of splicing in yeast cellular extracts and for
both steps of splicing in human cell extract. 38,39 These results suggested that
U2 and U6 are possibly the only snRNAs required for both steps of splicing,
and thus the only snRNAs that might be involved in splicing catalysis.
It has recently been shown that in spliceosomes assembled on pre-
mRNAs harboring a mutant 5 0 splice site, several positions within the
branch-binding sequence of the U2 snRNA can be mistakenly recognized
as the 5 0 splice site. This causes an aberrant trans -splicing in which the nucle-
otides 5 0 to the branch-binding sequence of the U2 snRNA are taken as an
exon and undergo both steps of splicing during which this region of U2
snRNA is ligated to the downstream exon in the pre-mRNA substrate. 40
These results are highly surprising since they imply that the branch-binding
site of U2 snRNA, which is treated as a substrate and is consumed in this
aberrant splicing reaction, is not essential for spliceosomal catalysis at least
under certain conditions. The branch-binding sequence of U2 is function-
ally the most critical region of the snRNA and the only part thought to be
positioned close to the active site, as the remainder of the RNA seems to
mainly fulfill structural roles by forming base-pairing interactions within
U2 or with U6. 15,17 Taken together, these results suggest that U6 snRNA
may be the only RNA that is absolutely crucial for splicing catalysis, at least
under the conditions studied so far.
Several additional lines of evidence suggest that U6, similar to its counter-
part domain V in group II introns, is part of the catalytic domain of the
spliceosome. Both U6 and domain V are highly conserved in primary
sequence and secondary structure, and the presence of two evolutionarily
nearly invariant sequences in U6, the ACAGAGA and the AGC boxes, sug-
gests a critical role in splicing for the snRNA ( Fig. 6.4 ). 15,17,24,41 Several mod-
ifications of the nucleobases or the backbone of these two regions (see below)
lead to either a complete or partial block to splicing, and some are interestingly
selective in blocking the second step of splicing. 42-47 In contrast, mutations
elsewhere in U6 or in other snRNAs often have a mild or no phenotype.
Crosslinking and mutational complementation analyses have indicated
that the first step of splicing occurs in close proximity to the ACAGAGA
box of U6, suggesting that the sequence is in the immediate vicinity of
or even forms part of the spliceosomal active site. 48,49 In the recently pub-
lished high-resolution structure of a self-splicing group II intron, 13,25 the
catalytic core is formed by the AGC triad and the asymmetric internal bulge
of domain V along with J2/3, a short purine-rich sequence that joins
domains II and III and is considered functionally equivalent

to the