The Role of snRNAs in Spliceosomal Catalysis - Progress in Molecular Biology and Translational Science - page 199

the spliceosomal catalytic core, U2 forms a number of base-paired helices
(helices I, II, and III) with U6 that are thought to act as scaffolds for juxta-
posing the reactive groups and the rest of the active site components
( Fig. 6.3 ). 15,17,41 While the details of the base-pairing interactions may be
slightly different in yeast and human spliceosomes, 53-55 the general architec-
ture of the U6/U2 base-paired complex is conserved. The invariant
ACAGAGA box of U6 and the branch-binding sequence of U2 are juxta-
posed via base-pairing interactions that flank the two sequences and form the
U6/U2 helix I and helix III in higher eukaryotes ( Fig. 6.3 ). This scaffold-
like base-paired structure also connects these two functionally critical
regions with the other invariant sequence of U6, the AGC triad, and the
ISL in U6 that contains a conserved, functionally required metal ion-binding
pocket 27,31 ( Figs. 6.3 and 6.4 ).
As described above (see also Fig. 6.2 ), the sequences in group II introns
that are equivalent to the U6 ACAGAGA and AGC boxes and the ISL metal
ion-binding pocket converge to form the active site in these ribozymes, and it
is thus conceivable that in the three-dimensional structure of the U6/U2
complex within activated spliceosomes these regions may be juxtaposed to
create a similar active site. Experimental evidence pointing to the possibility
of such a spatial arrangement was provided by hydroxyl radical footprinting
experiments in assembled spliceosomes both before and after the first step
of splicing. 56 In these studies, hydroxyl radicals generated by a tethered
Fe-BABE probe that was attached to the 10th nucleotide of the intron resulted
in cleavage of the nucleotides upstream of the ACAGAGA box in U6 and the
residues close to the metal ion-binding pocket in the U6 ISL ( Fig. 6.1 ), prov-
ing the proximity of these two structures in the folded U6/U2 complex both
before and after the first step of splicing. In addition, in vivo mutagenesis studies
have pointed to the presence of a long-range interaction between U2 residues
that are located across the helix from the AGC triad and the second G in the
ACAGAGA box ( Fig. 6.3 ), 57 which suggested the proximity of these two
invariant domains of U6 in the spliceosomes. Collectively, the existing data
suggest that the catalytically critical regions in U6 are positioned close to each
other in the folded, active structure of the U6/U2 complex found in the acti-
vated spliceosomes, perhaps in an arrangement similar or even identical to the
one found in group II introns. 12-14
The transition from the first step of splicing to the second by necessity
involves remodeling of the catalytic core, since at the very least the products
of the first step have to be removed and/or repositioned for the second step
of splicing to occur ( Fig. 6.1 ). 55,58-60 However, it is likely that the same