In vivo RNA structural probing of uracil and guanine base-pairing by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)

Abstract

Many biological functions performed by RNAs arise from their in vivo structures. The structure of the same RNA can differ in vitro and in vivo owing in part to the influence of molecules ranging from protons to secondary metabolites to proteins. Chemical reagents that modify the Watson-Crick (WC) face of unprotected RNA bases report on the absence of base-pairing and so are of value to determining structures adopted by RNAs. Reagents have thus been sought that can report on the native RNA structures that prevail in living cells. Dimethyl sulfate (DMS) and glyoxal penetrate cell membranes and inform on RNA secondary structure in vivo through modification of adenine (A), cytosine (C), and guanine (G) bases. Uracil (U) bases, however, have thus far eluded characterization in vivo. Herein, we show that the water-soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is capable of modifying the WC face of U and G in vivo, favoring the former nucleobase by a factor of ∼1.5, and doing so in the eukaryote rice, as well as in the Gram-negative bacterium Escherichia coli While both EDC and glyoxal target Gs, EDC reacts with Gs in their typical neutral state, while glyoxal requires Gs to populate the rare anionic state. EDC may thus be more generally useful; however, comparison of the reactivity of EDC and glyoxal may allow the identification of Gs with perturbed pK_as in vivo and genome-wide. Overall, use of EDC with DMS allows in vivo probing of the base-pairing status of all four RNA bases.

Keywords: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; EDC; RNA structure; in vivo RNA probing.

FIGURE 1.

In vitro modification of rice 5.8S rRNA by EDC analyzed by denaturing page of cDNAs after reverse transcription. (A) Reactions with the indicated EDC concentrations for 5 min. Dideoxy sequencing lanes, a control reaction lacking EDC, and reactions with EDC are shown. Blue text to the left indicates nucleotides within the sequence of the examined range of G53 to C143. (B) Reactive nucleotides in either 57 mM or 85 mM EDC mapped as hexagons and circles, respectively, onto the relevant portion of the rice 5.8S rRNA comparative structure. Colors indicate the level of modification for nucleotides exceeding the calculated significance value for which a base is considered modified (defined in Materials and Methods) after normalization and scaling such that all values fall between 0 and 1.

FIGURE 2.

Reaction scheme for base modification by EDC, shown in red. In the first step, EDC abstracts a proton from the endocyclic N3 of U. The resulting anionic lone pair on the nucleobase attacks the cationic carbodiimide moiety, leading to neutralization and covalent attachment of the EDC adduct to the base. EDC reacts with the endocyclic N1 of G in a similar fashion.

FIGURE 3.

In vitro EDC modification of rice 5.8S rRNA at various pH and EDC concentrations. (A) Denaturing PAGE analysis of cDNAs generated after RT. Reaction conditions at pH 7, pH 8, and pH 9.2 are shown along with dideoxy sequencing lanes. (B) Comparison of band intensities for all Us and Gs within the examined range of G55 to G138; reactions at 113 mM EDC are excluded due to excessive modification of the RNA. Colored boxes represent U or G modification above the calculated significance value (S); green boxes represent S to 3 S; yellow boxes represent >3 S to 6 S; orange boxes represent >6 S to 10 S; and dark red boxes represent >10 S. White boxes represent Us or Gs that are not significantly modified by EDC.

FIGURE 4.

In vivo EDC modification of rice 5.8S rRNA analyzed by denaturing PAGE of cDNAs after RT. (A) Reaction conditions at buffer pH 8 with 113 mM, 283 mM, and 565 mM EDC are shown along with dideoxy sequencing lanes. (B) Reaction conditions at buffer pH from 6 to 9.2 and at 113 mM or 283 mM EDC are shown along with dideoxy sequencing lanes. Reactions with 113 mM EDC at buffer pH 9.2 are shown twice, in lanes 12 and 13. (C) Reaction conditions at buffer pH 7 and 283 mM EDC with 2 min, 5 min, and 10 min durations are shown along with dideoxy sequencing lanes. The sequencing lanes were run on a different portion of the same gel as the experimental lanes, as indicated by the gray brackets.

FIGURE 5.

Comparison of in vivo EDC and phenylglyoxal modification of rice 5.8S and 28S rRNAs analyzed by denaturing PAGE of cDNAs after RT. (A) Comparison of EDC and phenylglyoxal (PG) modification of rice 5.8S rRNA under conditions where either a water wash (W) or 1 g of DTT (D) was used as a reaction quench, along with dideoxy sequencing lanes. Rice tissue not treated with reagent nor subjected to quenching is shown as NRT in lane 11. The three Gs modified by phenylglyoxal are indicated in purple text, while Gs modified by both EDC and phenylglyoxal are in red text. The section from C122 to C133 was run on a different portion of the same gel. (B) Nucleotides reactive with phenylglyoxal or EDC mapped as hexagons or circles, respectively, onto the relevant portion of rice 5.8S rRNA comparative structure. Colors indicate the level of modification after normalization and scaling such that all values fall between 0 and 1. The quench composition (water wash or DTT; see Supplemental Information) had no effect on observed EDC reactivity. (C) Comparison of EDC and phenylglyoxal modification of rice 28S rRNA. Conditions are the same as in panel A. (D) Nucleotides reactive with EDC or phenylglyoxal mapped onto the relevant portion of rice 28S rRNA comparative structure. Red discs indicate nucleotides modified solely by EDC while cyan discs indicate nucleotides modified by both EDC and phenylglyoxal. Data between 280 and 270 are omitted as too close to the primer, which ends at 280.

FIGURE 6.

In vivo EDC modification of E. coli 16S rRNA. (A) EDC concentration assays. Denaturing PAGE analysis of cDNAs generated after RT. Reactions in EDC from 28 mM to 85 mM are shown along with sequencing lanes. Blue text inset in the gel shows the true position of the sequence in relation to the experimental lanes, as part of the sequencing lanes were shifted by a crease in the gel. Red text indicates Gs and Us, while orange text indicates As and Cs. (B) Agarose gel analysis of rRNA extracted from E. coli after treatment with 28 mM to 113 mM EDC. (C) Lower EDC concentration trials. Denaturing PAGE analysis of cDNAs after RT. Reactions in EDC from 6 mM to 28 mM are shown along with sequencing lanes. Red text indicates modified nucleotides. (D) Nucleotides reactive with EDC mapped onto the relevant portion of E. coli 16S rRNA comparative structure. Arrows pointing to the reactive nucleotides show reactions in 17 mM, 23 mM, and 28 mM EDC in separate segments, with the 17 mM EDC segment located closest to the arrow head. The color within each segment indicates the relative extent of modification above the significance value (S).