The Role of snRNAs in Spliceosomal Catalysis - Progress in Molecular Biology and Translational Science - page 206

in vitro -assembled U2-U6 complex may contain a sufficiently large share of
the spliceosomal active site for it to be functionally active in isolation.
A number of attempts at investigating this possibility has been made using
the in vitro -assembled U2-U6 complex and various minimal splicing sub-
strates. In one case, a magnesium ion-dependent catalyzed reaction was
observed in which the designated branch site adenosine in a short substrate
was covalently linked to the G residue in the AGC domain of U6. While the
reaction chemistry was clearly distinct from splicing and highly inefficient, it
nonetheless depended on the catalytically critical ACAGAGA and AGC
sequences in U6 and the ability of the substrate to base pair to the
branch-binding site of U2 in a way that would leave the branch site aden-
osine in an unpaired, bulged conformation. 76,77 In another U6/U2-
catalyzed reaction, a covalent linkage was formed between the branch site
sequence of an intron-like short oligonucleotide substrate and a short
sequence resembling the 5 0 splice site consensus. This reaction, which
was also dependent on the two invariant U6 sequences, resulted in release
of a fragment of the 5 0 splice site-like sequence, reminiscent of the release of
the 5 0 exon after the first step of splicing. 78 In both these reactions, the low
efficiency of product formation prohibited a thorough analysis of the chem-
istry of the reaction and its requirements. However, the dependence of both
reactions on invariant sequences in U6 and the presence of magnesium ions
in the buffer indicated that at least to a limited extent they are related to
spliceosomal catalysis.
Recently, by modifying the positioning of short oligonucleotides used as
splicing substrates, it was shown that the in vitro -assembled human U2/U6
complex can indeed catalyze a two-step splicing reaction almost identical to
the one catalyzed by the self-splicing group II introns and the spliceosome. 79
In the first step of this reaction, the short oligonucleotide corresponding to
the 5 0 splice site was cleaved via a hydrolysis reaction resulting in the release
of an “exon” with a free 3 0 -OH group ( Fig. 6.6 ). The second step involved a
transesterification reaction between the newly released 3 0 -hydroxyl group of
the cleaved first exon and an internal phosphate in the 3 0 splice site substrate
resulting in the release of an “intronic” sequence and formation of a linear
product containing the “exonic” fragments of the two substrates joined
together by a 3 0 -5 0 phosphodiester linkage ( Fig. 6.6 ). In addition to per-
forming this two-step splicing reaction on a bipartite substrate, the human
U6/U2 complex was also able to catalyze the removal of an intervening
sequence from a short contiguous “mini-pre-mRNA” substrate, followed
by ligation of the “exonic” sequences into a linear product identical to