The Role of snRNAs in Spliceosomal Catalysis - Progress in Molecular Biology and Translational Science - page 204

complexes, 68 the bulged U residue in the U6 ISL is also part of a functionally
critical metal ion-binding pocket ( Fig. 6.4 ). 27,31,68,71 Phosphorothioate
interference studies in nuclear extracts have provided evidence for function-
ally important metal ion coordination by the nonbridging phosphate oxygen
located 5 0 to the bulged U residue. 31-33 Interestingly, NMR studies have
provided direct evidence for metal ion binding to this phosphate group
in isolated U6 ISLs, proving that the metal ion-binding ability was an inher-
ent property of the snRNA structure. 27,29 NMR and biochemical studies
have similarly demonstrated the existence of an analogously positioned
metal ion-binding pocket in the counterpart of the ISL in domain V of
group II introns 26,28 that helps coordinate the metal ions found near the
active site residues. 13,25 It is conceivable that the metal ion-binding pockets
in the U6 ISL and domain V may play a similar functional role in the two
systems.
In addition to the ISL, phosphorothioate interference studies in yeast and
nematode nuclear extracts have suggested the presence of two metal ion-
binding sites in U6 in the ACAGAGA and AGC boxes ( Fig. 6.4 ). 32,33 Anal-
ogous sequences in group II introns, together with the metal ion-binding
pocket in the asymmetric internal loop of domain V, contain the functional
groups involved in coordination of the catalytic metal ions in the putative
active site ( Fig. 6.2 ). 13,25 Using protein-free snRNAs, Yuan and col-
leagues 30 employed an innovative FRET-based assay that took advantage
of the increase in the luminescence of lanthanide ions (e.g., terbium(III)
ions) upon forming inner sphere coordination with a metal ion-binding site.
By monitoring changes in the luminescence of the site-bound Tb(III) ions
and FRET with a covalently attached fluorescent dye, three metal ion-
binding sites could be detected in the U6/U2 complex corresponding to
the ACAGAGA sequence, the vicinity of the AGC triad, and the asymmet-
ric bulge of the U6 ISL. These data provide direct evidence for metal ion
binding in the ACAGAGA and AGC sequences and suggest that the reduc-
tion in splicing efficiency observed upon phosphorothioate substitutions in
the ACAGAGA and AGC sequences in U6 may result from the loss of
functionally required inner sphere coordinated metal ions. Perhaps more
importantly, these studies show that in the absence of all other spliceosomal
factors, the complex of U6 and U2 snRNAs has the inherent ability to
coordinate metal ions at these functionally critical sequences.
Do these metal ion-binding sites indeed correspond to the functional
groups coordinating active site metal ions? Piccirilli and colleagues 21,23 rep-
laced the 3 0 -linked oxygen at either the 5 0 or the 3 0 splice site with sulfur and