Skip to main content
. 2020 May 20;6(21):eaaz2059. doi: 10.1126/sciadv.aaz2059

Fig. 1. Discovery of an endogenously expressed micropeptide encoded by MIR155HG.

Fig. 1

(A) Schematic representation of P155 translation. Human MIR155HG spans 13,024 bp and has three exons. P155 is translated by ORF1 (indicated by yellow boxes), which comprises the end of exon 2 and the head of exon 3. The nucleotide and amino acid sequences of ORF1 are highlighted in red and the pre–miR-155 is indicated by a bluish color. (B) Schematic representation of P155 EGFP knock-in strategy. The EGFP (without its own ATG) was inserted after the last coding codon (GTT-valine) of P155 by CRISPR/Cas9-mediated homologous recombination in HEK293T cells. The front homologous arm is a 501-bp fragment before the termination codon of P155 sequence and the back homologous arm is a 501-bp fragment starting with the P155 termination codon, E3: exon 3. (C) PCR detection of EGFP knock-in efficiency. Target band is indicated by the yellow box. (D) Fluorescence imaging of P155-EGFP fusion protein expression. (E) Immunoblotting verification of P155-EGFP fusion protein in HEK293T cells. Protein lysate of EGFP plasmid–transfected HEK293T cells served as a negative control. The target band is indicated by black arrowheads, and the EGFP location is visible as a black line. (F) Immunoblotting detection of endogenously expressed P155 in human moDCs with P155-specific antibody pre-enrichment. Chemically synthesized P155 served as a positive control, and the target band is indicated by the black arrowheads. (G) LC-MS verification of the P155 endogenous expression in OCI-LY-1 cells with P155-specific antibody pre-enrichment. Scale bar, 100 μm. Data (D to F) are representative of three independent experiments. Photo credit: Liman Niu (Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine).