Solution Structure of Poly(UG) RNA

Abstract

Poly(UG) or "pUG" RNAs are UG or GU dinucleotide repeat sequences which are highly abundant in eukaryotes. Post-transcriptional addition of pUGs to RNA 3' ends marks mRNAs as vectors for gene silencing in C. elegans. We previously determined the crystal structure of pUG RNA bound to the ligand N-methyl mesoporphyrin IX (NMM), but the structure of free pUG RNA is unknown. Here we report the solution structure of the free pUG RNA (GU)₁₂, as determined by nuclear magnetic resonance spectroscopy and small and wide-angle x-ray scattering (NMR-SAXS-WAXS). The low complexity sequence and 4-fold symmetry of the structure result in overlapped NMR signals that complicate chemical shift assignment. We therefore utilized single site-specific deoxyribose modifications which did not perturb the structure and introduced well-resolved methylene signals that are easily identified in NMR spectra. The solution structure ensemble has a root mean squared deviation (RMSD) of 0.62 Å and is a compact, left-handed quadruplex with a Z-form backbone, or "pUG fold." Overall, the structure agrees with the crystal structure of (GU)₁₂ bound to NMM, indicating the pUG fold is unaltered by docking of the NMM ligand. The solution structure reveals conformational details that could not be resolved by x-ray crystallography, which explain how the pUG fold can form within longer RNAs.

Keywords: NMR; Quadruplex; RNA; SAXS; poly(UG).

Figure 1.

The pUG fold has four-fold symmetry of in solution. (A) Sequence of (GU)₁₂, one of the RNAs used in this study. (B) Crystal structure of (GU)_11.5 bound to N-methyl mesoporphyrin IX (NMM)(red), PDB ID 7MKT. Purple spheres are K⁺ ions. (C) Short-hand description (50) of the pUG-fold observed in the crystal structure bound to NMM. Upper case nucleotides are Anti, lower case are Syn. Bold nucleotides are 2’-endo and italicized nucleotides are 3’ endo. Inverted nucleotides indicate inverted strand polarity relative to the 5’ nucleotide. < indicates bulged uridines. Circled nucleotides are single nucleotide loops and the numbers shown refer to the nucleotide number in the sequence rather than the length of the loops (50). (D) ³¹P 1D NMR spectrum showing six main peaks for the 24-nucleotide (GU)₁₂ RNA. Resonance assignments are indicated above the peaks. Labels are color coded to match the RNA structure with G quartet nucleotides in green, U nucleotides in blue. The two small unassigned peaks are likely due to the presence of a small amount of RNA that is longer by one nucleotide (n+1), a common byproduct of run-off transcription by T7 RNA polymerase. Note there is no assignment for G1 because the 5’ end has a hydroxyl group. (E) 2D ¹H,¹³C HSQC of aromatic resonances. The spectrum shown is an overlay of two experiments, one performed on a G-labeled sample (green) and one from a U-labeled sample (blue). The terminal U24 nucleotide has two chemical shifts (24a and 24b) owing to a minor population of n+1 transcription product. (F). 2D ¹H,¹³C HSQC of ribose resonances for the G-labeled sample and (G) U-labeled sample.

Figure 2.

Ribose to nucleobase correlations via through-bond triple resonance HCN experiments. (A) G-labeled ribose to aromatic correlations via the N9 glycosidic nitrogen. (B). U-labeled ribose to aromatic correlations via the N1 glycosidic nitrogen. Connectivity to aromatic resonances for U4,10,16,22 and U24b are observed only at lower contour levels due to low signal intensities.

Figure 3.

2D ¹H,¹H-NOESY of (GU)₁₂ single deoxyribose variants. Top rows show intra-residue NOE cross-peaks between aromatic and H2’ and H2” methylene protons from 2’-deoxyribose modifications at positions G1, U2, G3, U4, G5 and U6. Bottom row shows overlay with NOESY spectrum of wild type (GU)₁₂ (black), with red arrows showing chemical shift perturbation by the deoxy substitutions.

Figure 4.

(A) 2D ¹H,¹⁵N-HSQC of imino signals from G-labelled and (B) U-labeled (GU)₁₂ RNA. (C) 2D ¹H, ¹H ¹⁵N-HSQC-NOESY of U-labeled sample. (D and E). 2D planes of 3D ¹³C-filtered HSQC-NOESY spectra showing inter-residue NOEs between U4 and G5 observed using the G-labeled (D) and U-labeled (E) samples.

Figure 5.

(GU)₁₂ structure ensemble. (A) The 20 lowest energy structure models for (GU)₁₂ are shown. (B) Energy minimized average structure, as viewed from above the G1-G7-G13-G19 quartet. (C) Same as (B) but rotated by 90°. (D) SAXS-WAXS profiles. Experimental x-ray scattering intensities as a function of the scattering vector q are shown in black. Predicted X-ray scattering profiles back-calculated from the (GU)₁₂ NMR-SAXS-WAXS structures in (A) were computed using Crysol (red) and Xplor-NIH software (blue). The predicted scattering profiles from the crystal structure 7MKT, minus the NMM ligand, were calculated using Crysol (green). (E) (GU)₁₂ electron density model calculated from the SAXS-WAXS data using DENSS algorithm with 4-fold symmetry applied. Image was prepared using Pymol using the default DENSS color gradient (28) corresponding to standard units of electron density (sigma) where blue is 2.5, cyan is 5, green is 7.5, yellow is 10 and red is 15–200. (F) same as (E) but rotated 90°.

Figure 6.

1D ³¹P NMR spectra comparing (GU)₁₂, the final 27 nucleotides of the 3’ end of the oma-1 mRNA (oma-1), and the same oma-1 sequence followed by a (UG)₁₂ pUG tail (oma-1-pUG). The 51-nucleotide oma-1-pUG spectrum shows two distinct pUG fold peaks at 0.5 and −3 ppm which arise from U2, U8, U14, U20 and G5, G11, G17, G23 as indicated.

Figure 7.

Comparison of pUG fold to Z RNA. (A) A segment of (GU)₁₂. (B) A single strand segment of duplex Z-RNA (PDB 2GXB). Ribose orientations are indicated with black arrows. Conformational similarities are listed in green text beside the corresponding nucleotides. (C and D) Top and side views of the first two stacked nucleotides of each structure. A lone pair-pi interaction in Z-RNA is shown as a dashed line (D); this interaction is replaced by base stacking in the pUG fold.