Minimum-Energy Path for a U6 RNA Conformational Change Involving Protonation, Base-Pair Rearrangement and Base Flipping

Abstract

The U6 RNA internal stem–loop (U6 ISL) is a highly conserved domain of the spliceosome that is important for pre-mRNA splicing. The U6 ISL contains an internal loop that is in equilibrium between two conformations controlled by the protonation state of an adenine (pK_a = 6.5). Lower pH favors formation of a protonated C–A⁺ wobble pair and base flipping of the adjacent uracil. Higher pH favors stacking of the uracil and allows an essential metal ion to bind at this position. Here, we define the minimal-energy path for this conformational transition. To do this, we solved the U6 ISL structure at higher pH (8.0) in order to eliminate interference from the low-pH conformer. This structure reveals disruption of the protonated C–A⁺ pair and formation of a new C–U pair, which explains the preference for a stacked uracil at higher pH. Next, we used nudged elastic band molecular dynamics simulations to calculate the minimum-energy path between the two conformations. Our results indicate that the C–U pair is dynamic, which allows formation of the more stable C–A⁺ pair upon adenine protonation. After formation of the C–A⁺ pair, the unpaired uracil follows a minor-groove base-flipping pathway. Molecular dynamics simulations suggest that the extrahelical uracil is stabilized by contacts with the adjacent helix.

Introduction

U6 RNA is one of five snRNAs (U1, U2, U4, U5 and U6) that, along with more than 70 proteins, assemble on the pre-mRNA to form a large and dynamic complex called the spliceosome.1, 2 The spliceosome catalyzes precise excision of introns and ligation of the flanking exons through a two-step phosphotransesterification reaction resulting in production of mature mRNA. Once fully assembled, the spliceosome is activated through a number of structural rearrangements in the snRNAs, facilitated by protein components.³ These rearrangements result in disruption of the U4-U6 complex and formation of the U2-U6 complex (Fig. 1), which assists in splicing catalysis through substrate positioning and possibly chemistry.⁴ The association of U2 and U6 leads to formation of a highly conserved U6 internal stem–loop (U6 ISL).³

The secondary structure of the yeast U6 RNA was investigated by in vivo and in vitro dimethyl sulfate probing experiments, and these data are consistent with the presence of an asymmetric 1 × 2 internal loop in the center of the U6 ISL⁵ (Fig. 1). This highly conserved internal loop seems to be crucial for spliceosome function, since it coordinates an essential Mg²⁺ ion at the U80 position,6, 7 mutations inside the bulge sequence generate growth defects that are not fully suppressed by compensatory mutations in U4 RNA,⁸ and hydroxyl radical probing experiments place the human ISL internal loop in the vicinity of the 5′ splice site in activated spliceosomes.⁹ In addition, the internal-loop sequence found in the U6 ISL is also found in other RNAs. A database survey of 955 different RNA secondary structures found 12 occurrences of this 1 × 2 internal loop in 16S and 23S rRNAs and in RNase P.¹⁰

The structure and dynamics of the U6 ISL have been investigated by NMR spectroscopy.7, 11, 12, 13, 14, 15, 16 Consistent with the dimethyl sulfate probing results, the NMR solution structure determined at pH 7.0 shows that the U6 ISL contains a partially protonated C67–A79 wobble base pair and an unpaired U80 that is stacked in the helix.7, 11 In addition, comparison of the ISL structures at pH 7.0 and pH 5.7 indicates that the U80 nucleotide is in equilibrium between a stacked and a flipped-out conformation, with the latter predominating at low pH.¹² The pH-dependent conformational switch appears to be induced by protonation of the A79 N1 atom (pK_a = 6.5), which results in stabilization of the C67–A79 wobble pair and base-flipping of the neighboring U80 nucleotide.¹² Analyses of NMR relaxation rates indicate that the lifetime of the protonated C67–A79 N1 base pair is 20 μs,¹² while base-flipping of U80 occurs on the 80-μs time scale.¹³ Magnesium binding studies indicate that low pH inhibits metal ion binding, and the amount of metal ion binding is proportional to the fractional population of the stacked U80 (high pH) conformation.7, 13 Thus, the U6 ISL conformational equilibrium has the potential to regulate spliceosome activity.12, 13 However, the mechanism and associated energetics of this conformational transition have not been described, and, in particular, it is not clear why stacking of U80 is only favored upon disruption of the protonated C–A wobble pair. Although RNA is widely considered to be a dynamic molecule, the driving forces for structural rearrangements in RNA are poorly understood in general.

Since the population of protonated A79 is still significant at pH 7.0 (∼ 24%), we hypothesized that the pH 7.0 U6 ISL NMR structure is affected by conformational averaging due to interference from the low-pH conformation. If this hypothesis is true, then a high-pH structure should reveal a different conformation of the U6 ISL internal loop. Here we determine the NMR structure of U6 ISL at pH 8.0, and indeed this NMR structure reveals a different loop conformation with a C67–U80 pair and an unpaired A79. The previously determined low-pH (pH 5.7) structure and the pH 8.0 NMR structures were used as endpoints for computation of the minimum-energy path (MEP) for the conformational change using nudged elastic band (NEB) sampling.¹⁷ The results explain the physical basis for this conformational change, which involves a complex interplay of nucleobase protonation, alternative base-pair formation and base flipping.