Fig 1: MiR-342-5p and miR-608 directly targeted NAA10 for degradationA., B. The mutant NAA10-3′-UTR luciferase vectors were constructed by mutating the conserved sequences targeted by miR-342-5p and miR-608 in the NAA10-3′-UTR luciferase vector as indicated. C. Sequence aliments across species in the 3′-UTR of NAA10. D., E. wild-type or mutant NAA10-3′-UTR luciferase vectors were co-transfected with the indicated miRNA mimics or ASOs into SW620 D. and SW480 E. cells, and the luciferase activities were measured 24 h later. Experiments were repeated at least three independent times. Values represent the mean±s.d. *P < 0.05, calculated using Student's t-test.
Fig 2: Characterization of Naa12.(A) In vitro N-terminal acetyltransferase radioactive-based assay. Comparison of mouse Naa10 and Naa12 towards Naa10 peptide substrates, beta-actin (DDDIA-) and gamma-actin (EEEIA-), and the optimal NatA complex peptide substrate, SESSS-. Background control reactions were performed in the absence of either peptide or enzyme. Assays were performed in triplicate; error bars represent SEM. (B) Co-immunoprecipitation assay. HEK293 cells were transfected as indicated and lysed after 48 hr. Cell lysates were incubated with 1 µg anti-V5 antibody to precipitate V5-tagged Naa15. The isolated complexes were separated on SDS-PAGE and probed with the indicated antibodies. (C) Recombinant mouse Naa12/human Naa15 chimera complex activity. Radioactive acetyltransferase activity assay evaluating the activity of mNaa12-hNaa15 towards peptide (closed circles, ‘mNaa12-hNaa15’) and peptide chemical acetylation in the absence of enzyme (closed circles, ‘Buffer’) as well as chemical acetylation of the enzyme in the absence of peptide (open circles) assay and background (open circles). Error bars represent SD of two technical replicates. These are the same results from fraction #14 (both SESSS- and No Peptide) and both Buffer and Background used to illustrate the size-exclusion-purified mNaa12-hNaa15 complex activity in Figure 4—figure supplement 1F. Figure 4—source data 1.Characterization of a potential Naa10 homolog.
Fig 3: Selection of miRNAs targeting NAA10A. Bioinformatics analysis predicted miRNAs regulating NAA10 are listed below. B. Luciferase activities in SW620 cells were measured 24 h after cotransfection of the candidate miRNAs and a NAA10-3′-UTR-containing luciferase vector. C., D. SW480 or SW620 cells were transfected with the indicated miRNA mimics or anti-sense oligonucleotides (ASO), and 24 h later, NAA10 mRNA was extracted and analyzed by real-time PCR. E., F., G., H. Cells were transfected as in C. and D. and lysed 36 h later to collect whole cell lysates (WCL) that were subjected to western-blot (WB) analysis. Experiments were repeated at least three independent times. Values represent the mean±s.d. *P < 0.05, calculated using Student's t-test.
Fig 4: NAA10 restoration rescued miR-342-5p and miR-608 mediated cell proliferation, migration, and colony formation defects and promoted cell apoptosis suppressed by miR-342-5p or miR-608SW480 or SW620 cells were co-transfected with the indicated miRNA mimics and vectors. A. 36 h after transfection, cells were lysed and WCL were subjected to WB analysis (β-actin as a loading control). B., E. Single cells were seeded into 6-well plates 6 h after transfection, and the colonies were counted 24 h later. C., F. SW480 (upper panel) and SW620 cells were seeded into the upper part of a transwell chamber before transfection, and 24 h later cells in the bottom were counted under a microscope after staining. D., G. SW480 and SW620 cells were collected 24 h after transfection and then subjected to flow cytometry analysis. Experiments were repeated at least three independent times. Values represent the mean±s.d. *P < 0.05, calculated using Student's t-test.
Fig 5: Lethality in Naa10 Naa12 double-knockout (DKO) mice.Naa10 Naa12 DKO exhibit embryonic lethality. Pedigree and genotypes of pups and embryos at E10.5 and E18.5 from Naa10+/- Naa12+/- female mice crossed to the Naa10+/Y Naa12+/- male mice.
Supplier Page from Abcam for Anti-ARD1A antibody