Immunoprecipitation of endogenous modified RNAs. (A) Overview of the workflow for cross-linking modification-specific antibodies to endogenous RNAs. Fragmented total RNA from HEK293T cells was cross-linked to the antibody of interest using 254 nm UV light. RNA was immunoprecipitated, radiolabeled, and the RNA-antibody complexes were analyzed by SDS-PAGE. As a negative control, a nonirradiated setup was used. (B) Autoradiographs of the cross-linking experiments described in A using m6A antibody clones 9B7, Synaptic Systems (I), m5C antibody clone 32E2 and the commercial antibody 33D3 (Diagenode) (II), α-Ψ antibody clone 27C8 (III) and m26A antibody clone 60G3 (IV). (C) Binding competition assays between endogenous RNAs and free nucleosides. Specific antibodies were used for immunoprecipitation from 2 µg total HEK293T cell RNA and 75 ng GFP mRNA as a negative control spike-in. To test for specificity, 100 µM of the respective modified (e.g., m5C) and unmodified nucleoside (e.g., C) were added to the immunoprecipitation as competitor. The immunoprecipitated RNA was separated on an RNA gel and blotted onto a nylon membrane. A probe against the 18S or 5.8S rRNA and a probe against the GFP mRNA were used for detection. (D) Northern blots for endogenous 18S and 5.8S rRNAs as well as GFP mRNA spike-ins. The antibodies α-m6A 9B7, α-m5C 32E2, α-Ψ 27C8, and α-m26A 60G3 were used. Input samples (200 ng total RNA and 75 ng GFP mRNA) are shown in lanes 1 and immunoprecipitates using an IgG control antibody used as a negative control in lanes 2. The upper panels show the signals of the 18S/5.8S rRNA; the lower ones for the spiked-in a GFP mRNA. Lanes 3 show signals for untreated IP experiments, lanes 4 depict the signals of an IP competed with modified and lanes 5 with unmodified nucleosides. (E) Competition titration experiment of antibodies α-m6A 9B7, α-m5C 32E2, and α-Ψ 27C8. Different concentrations ranging from 10 to 100 µM of modified and unmodified nucleosides were added to RNA-IP experiments. For detection, probes against 18S and 5.8S rRNA were used. (F) Nucleoside-mediated elution of antibody-bound endogenous 18S rRNA. (G) Northern blots of immunoprecipitation experiments using antibodies α-m6A 9B7 and α-m5C 32E2. RNA either extracted from beads (lanes 2–5) or from specifically eluted supernatants (lanes 6–9) were analyzed by northern blotting. Lane 1 shows the input of the immunoprecipitation experiments.
