The U2/U6 complex lies at the heart of the catalytic core of the eukaryotic spliceosome, but its structure has been highly debated. Here, we have used smFRET to show that this important RNA complex acts as a Mg2+
-dependent conformational switch that can adopt at least three distinct conformations. This supports the hypothesis that U2/U6 adopts multiple conformations at various splicing stages,21
which may or may not reflect the presence of unique active sites for each step.35
In the high FRET conformation (N), the AGC triad extends the ISL as predicted for the 4-helix structure,19
and the ACAGAGA loop and U80 in the ISL are brought in close proximity by a stabilizing tertiary contact. Because of the expected relationship between these three regions during the first splicing step, it is possible that this conformation resembles the active conformation. The recent Group II intron ribozyme crystal structure provides a framework for this scenario (Supplementary Figure 8
. In this structure, C358
(proposed equivalents of the AGC triad) are clearly base paired with G383
, extending domain 5 (proposed equivalent of the U6 ISL). The C360–G383 base pair forms a triple with C377 (proposed equivalent of U80), which in turn stacks on G288 from J2/3 (proposed equivalent of the ACAGAGA loop). G288 also forms a triple with G359 and U384. Our U80 mutant results support the idea that these interactions also take place in the high FRET conformation of the U2/U6 complex. In the crystal structure, O2 in U80 is a hydrogen bond acceptor in the base triple. The same O2 in U80C and N3 or N7 in U80A could play a similar role in the mutants.
In the mid FRET conformation, the AGC triad still forms the extended ISL (4-helix structure),19
but the tertiary contact between the ACAGAGA loop and U80 is no longer formed, as its stability does not depend on the U80 mutations. Our current data, however, do not allow us to conclude whether this conformation plays a more direct role in splicing.
In the low FRET conformation, a junction migration takes place, resulting in the formation of helix IB (3-helix junction).16
The U80 mutants do not affect its stability, indicating that the ACAGAGA loop and U80 are not in direct contact. A recent paper has shown that helix I is important for both steps of splicing and that a conformational rearrangement must precede each step.18
This result is in agreement with our mutational data that link the formation of the low FRET conformation to an activating step between the first and second splicing steps. It is tempting to hypothesize that the observed structural dynamics also play an activating role in the first splicing step, however the mutations tested here do not provide enough support for this conclusion.
The role of Mg2+
ions at different stages of spliceosomal activation and catalysis has been established by experiments using phosphorothioate substituted RNAs.25,26,36,37
A key sensitive nucleotide is the phylogenetically conserved U80, which suggests either that this base plays a direct role in catalysis or is at least involved in a closely related step.26
Phosphorothioate substitutions in the 5' and 3' splice sites also involve Mg2+
ions in both splicing steps by activating the nuc-leophilic attack and stabilizing the leaving groups,25,36
and suggest the presence of a Mg2+
-dependent conformational change specific to the second step of splicing.36,37
We propose that this Mg2+
-induced conformational change corresponds to the one observed here. Based on this, however, one might expect an inverse effect, whereby Mg2+
ions would preferentially stabilize the high FRET structure. In the presence of essentially a constant supply of Mg2+
ions in vivo
, it is possible that the role of at least some of the spliceosomal proteins is to adjust the relative stability of these conformations to time the U2/U6 structural dynamics for accurate and efficient splicing.
We removed the highly conserved U6 ISL loop, but in accordance with a previous genetic study, in which the ISL was extended by one base pair, we do not expect this loop to significantly affect the structure of U2/U6 in vitro
Nonetheless, we have tested the effect of this deletion on the U2/U6 structural dynamics using a two-strand construct (Supplementary Figure 9
). Our results show that the two constructs behave almost identically. The three-strand construct, however, offers greater efficiency of synthesis, labeling and purification, and will facilitate future mutational studies.
We have recently elucidated the folding pathway of a self-splicing group II intron ribozyme, which has structural and catalytic similarities to the spliceosome.13
Its pathway also involves obligatory intermediates and is dominated by a Mg2+
capture step that activates junction dynamics for catalysis. The similarities between the U2/U6 structural dynamics and those of the group II intron ribozyme now expand the parallels between these two enzymes and suggest the existence of evolutionarily conserved structural dynamics.