The Role of snRNAs in Spliceosomal Catalysis - Progress in Molecular Biology and Translational Science - page 193

unusual 2 0 -5 0 linkage between the branch site adenosine and the first nucle-
otide of the intron ( Fig. 6.1 ). During the second step, the free 3 0 -hydroxyl
moiety of the newly released exon is activated for a similar nucleophilic
attack on the 3 0 splice site, resulting in ligation of the two exons and release
of the intron as a branched lariat. 5,17 Both group II intron and spliceosomal
splicing reactions are dependent on divalent cations, and phosphorothioate
substitution analyses at the splice sites have revealed an almost identical inter-
ference pattern (see below). The latter could result from a similar arrange-
ment of catalytically essential metal ions in the active site of each system. 18-23
In addition to these intriguing parallels, significant similarities between
the sequence and secondary structure of the snRNAs and several domains
of group II introns have led to the hypothesis that the snRNAs are an evo-
lutionary descendant of group II-like introns, and thus they might have a
catalytic role in the spliceosomes. Group II introns have a conserved second-
ary structure consisting of six domains, named domains I through VI, and
they fold into a complex tertiary structure. 13,24,25 The structure of the cat-
alytically critical domain V, the most conserved domain of group II introns,
closely resembles an intramolecular stem-loop (ISL) in U6 snRNA in its
overall structure and primary sequence ( Fig. 6.2 ). Both structures contain
two short base-paired helices separated by an asymmetric bulge that forms
a metal ion-binding pocket, 26-31 and both terminate in a GNRA-type tetra-
or pentaloop. Further, both contain a catalytically essential AGC triad at
the 5 0 end that interacts with functionally essential divalent cations in
group II introns and likely does so in the spliceosome. 25,30,32-34 Interest-
ingly, domain V of a group II intron could replace the ISL of U6 in an
in vivo splicing assay, 35 further strengthening the functional equivalence
of the two structures and bolstering the possibility of an evolutionary rela-
tionship between them.
Another domain of group II introns that has a direct counterpart in the
spliceosomes is domain VI, which contains the equivalent of the base-paired
structure formed between U2 snRNA and the branch site of introns
( Figs. 6.1-6.3 ). In both cases, the branch site adenosine which carries the
nucleophile for the first step of splicing is constrained in a bulged conforma-
tion required for optimal activation. 17,24,36 In addition, at least three
sequences in domain I (EBS1, EBS2, and EBS3; Fig. 6.2 ) bind the exonic
sequences in a manner that resembles how U5 snRNA binds to and aligns
the two exons for the second step of splicing. Domain swapping experiments
in which a fragment of domain I was replaced by U5 further demonstrated
their functional equivalence. 24,37 Yet another subdomain of domain I ( e 0 in