The Role of snRNAs in Spliceosomal Catalysis - Progress in Molecular Biology and Translational Science - page 201

mentioned above, the invariant ACAGAGA box of U6 fits both these
criteria. Briefly, several point mutations in this conserved region are incom-
patible with splicing or result in a block to splicing after the first step. 42-47 In
addition, UV crosslinking and complementation studies have proved the
proximity and the presence of a base-pairing interaction between the 5 0
splice site and the last nucleotides of the ACAG AGA box
( Fig. 6.3 ). 48,49,61,64 Finally, phosphorothioate substitution analyses of the
backbone phosphate groups in U6 snRNA in yeast and nematodes have
identified a number of phosphate oxygens that when substituted with sulfur
result in a block to the first or second step of splicing ( Fig. 6.4 ). 15,32,33
Intriguingly, all of the phosphorothioate interference sites fall within the
ACAGAGA and AGC sequences and the asymmetric bulge of the ISL.
The close similarities between the phosphorothioate interference pattern
in U6 and that in domain V of group II introns further adds to the parallels
between the two systems ( Fig. 6.4 ). 16,24
Similar to the ACAGAGA box, the invariant AGC triad of U6 is also
highly sensitive to modifications of nucleobases and backbone
elements, 54,65 and data from group II introns suggest a direct catalytic role
for the middle G residue in that system. 34,66 As mentioned above, in vivo
complementation studies in yeast suggest that in the tertiary structure of
the U6/U2 complex, the AGC triad is positioned in spatial proximity to
the ACAGAGA box ( Fig. 6.3 ). 57 This observation has been confirmed
by several biochemical and structural biology studies in protein-free
snRNAs. Initially, it was shown that in vitro -transcribed, protein-free RNAs
corresponding to the functionally required domains of human U2 and U6
snRNAs can efficiently form a base-paired complex in vitro in the presence of
magnesium ions. 67 Psoralen-mediated crosslinking analyses have also indi-
cated the formation of U6/U2 helices I, II, and III in this base-paired com-
plex ( Fig. 6.3 , Valadkhan, unpublished data). In addition, crosslinking
studies indicated that the interaction described above between the second
G residue in the ACAGAGA box and the U2 sequence across the helix from
the AGC triad 57 ( Fig. 6.3 ) is also present in this protein-free, in vitro -
assembled complex. 67 Recently, fluorescence resonance energy transfer
(FRET) experiments have shown the presence of at least three different
folded structures for the U6/U2 base-paired complex in solution. 68 Inter-
estingly, in one of these structures the ACAGAGA-containing stem and
the U6 ISL are positioned in close proximity, similar to the arrangement pre-
viously shown to exist in activated spliceosomes. 56 It was also shown that the
conserved, bulged U residue in the U6 ISL is required for the formation of