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. 2022 Sep 26;50(18):10695–10716. doi: 10.1093/nar/gkac817

Figure 1.

Figure 1.

Human NOP2/NSUN1 miCLIP design. (A) Alignment of the protein sequences of human NSUN family members showing conserved cysteine residues (highlighted) required for catalytic activity (C459 and C513). The cysteine mutated for the miCLIP assay is C459 in motif IV. (B) Cartoon depicting that only the NOP2/NUSN1 C459A mutant forms irreversible covalent crosslinks with RNA substrates. (C) FLAG-tagged NOP2/NSUN1 Wildtype (WT) and C459A mutant were expressed in HEK293T cells and immunoprecipitated with a FLAG antibody. Immuno-precipitated proteins were detected by Western Blotting using a FLAG antibody. (D) FLAG-tagged NOP2/NSUN1 WT and C459A mutant expressed in HEK293T cells were immunoprecipitated with a FLAG antibody. Co-precipitated RNA was 3′-end ligated with pCp-biotin for visualization. After SDS-PAGE separation and membrane transfer, the pCp-biotin-labeled RNA was detected with Streptavidin-IR800. Immunoprecipitated NOP2/NSUN1 WT and C459A mutant were detected by immunoblotting with a FLAG antibody. The right panel shows a high exposure of the pCp-biotin-labeled RNA with relative densitometry analysis. (E) Schematic overview of the NOP2/NSUN1 miCLIP-sequencing. HEK293T cells expressing NOP2/NSUN1 WT and C459A mutant were lysed, digested with RNase, immunoprecipitated with FLAG antibody, separated on SDS-PAGE and transferred to membrane. NOP2/NSUN1 WT and C459A mutant-associated RNAs and their respective size-matched inputs (SMInputs) were extracted from the membrane and processed for libraries and Illumina sequencing.