Identification of recognition residues for ligation-based detection and quantitation of pseudouridine and N6-methyladenosine

Abstract

Over 100 chemical types of RNA modifications have been identified in thousands of sites in all three domains of life. Recent data suggest that modifications function synergistically to mediate biological function, and that cells may coordinately modulate modification levels for regulatory purposes. However, this area of RNA biology remains largely unexplored due to the lack of robust, high-throughput methods to quantify the extent of modification at specific sites. Recently, we developed a facile enzymatic ligation-based method for detection and quantitation of methylated 2'-hydroxyl groups within RNA. Here we exploit the principles of molecular recognition and nucleic acid chemistry to establish the experimental parameters for ligation-based detection and quantitation of pseudouridine (Psi) and N6-methyladenosine (m6A), two abundant modifications in eukaryotic rRNA/tRNA and mRNA, respectively. Detection of pseudouridylation at several sites in the large subunit rRNA derived from yeast demonstrates the feasibility of the approach for analysis of pseudouridylation in biological RNA samples.

Figure 1.

(A) Chemical structures of Ψ and m⁶A. (B) Scheme for T4 DNA ligase-catalyzed joining of two DNA substrates. In the ternary RNA/DNA complex, the black line corresponds to the 30-mer RNA template with the modified nucleotide (open circle) located at the 15th position. Blue lines correspond to the ligation substrates with the recognition residue shown as a filled blue circle.

Figure 2.

Identifying a recognition residue for Ψ. (A) Within pseudouridine-containing RNA duplexes, a water molecule localizes in the major groove, forming hydrogen bonds to the phosphate backbone and to Ψ via N¹H (25–27). (B) Synthetic scheme for N⁶-aryldeoxyadenosine analogs. (C) Relative efficiency for U versus Ψ-directed ligation correlates with the hydrophobicity of the recognition residue. The yield of ligation product using the unmodified 30-mer RNA template (U15) relative to the yield of ligation product using the Ψ 15 template gives the ‘fold discrimination’ plotted on the y-axis. Log P values (x-axis) for the deoxyadenosine substituent at the recognition position were calculated using CS Chem 3D (version 5.0). The 3′ nucleotide at the ligation junction bears at the 2′-position either a hydrogen atom (dC-anchor), a methoxyl group (Cm-anchor) or a hydroxyl group (rC-anchor). (D) Molecular model of the base pair N⁶-phenanthren-9-yl-A: Ψ in an A-form helix. The phenanthrene ring is in green and the N¹H-coordinated water is in purple. (E) Ligation yield correlates linearly with the fraction of pseudouridylation f(Ψ). Ligation reactions contained U and Ψ 30-mer RNA templates mixed together in defined ratios as indicated. Linear fit has a r-value of 0.998 and P-value of <0.0001.

Figure 3.

Identifying a recognition residue for m⁶A. (A) The sheared G–A base pair found in many RNA structures (left). Within this purine–purine base pair N⁶H(A) and 2′OH(G) reside in close proximity to each other. The methyl group of m⁶A (right) might sterically clash with the phosphate backbone of G. (B) Synthetic scheme for N⁶-methyl-rA phosphoramidite. (C) Efficiency of A and m⁶A-directed ligation using different recognition residues. The 30-mer RNA templates contained at position 15 either unmodified adenosine (A) or N⁶-methyladenosine (m⁶A). Substrates contained one of the following recognition residues: dG (2′-deoxyguanosine), Gm (2′-methoxyguanosine), dT (thymidine) or Um (2′-methoxyuridine). (D) Ligation yield correlates linearly with the fraction of adenosine methylation f(m⁶A). Ligation reactions contained adenosine and N⁶-methyladenosine 30-mer RNA templates mixed together in defined ratios as indicated. Linear fit has a r-value of 0.964 and P-value of 0.002.

Figure 4.

Detecting changes in pseudouridylation levels within ribosomal RNA from yeast. (A) Ligation reactions detect the absence of 1051 pseudouridylation in an snR81 deletion strain. The small nucleolar RNA snR81 specifically guides the pseudouridylation at U1051 in the 3393 nt 25S rRNA from yeast. Ligation reactions contained 30-mer RNA template (std), RNA derived either from a wild-type yeast strain or from the corresponding ▵snR81 deletion strain, and substrates containing N⁶-phenanthren-9-yl-adenosine in the recognition position designed for ligations at residues 1041, 1051 and 2314 of rRNA and residue 15 of the 30-mer RNA template. (B) Quantitative differences in the amount of ligation products for U/Ψ1051 in rRNA. The two-sided t-test P-value for these data is 0.0885. (C) Ligations as described in (A) but using oligonucleotides substrates containing adenosine at the recognition position.