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Epitranscriptomic profiling across cell types reveals associations between APOBEC1-mediated RNA editing, gene expression outcomes, and cellular function

  1. F. Nina Papavasilioua,h,3
  1. aLaboratory of Lymphocyte Biology, The Rockefeller University, New York, NY 10065;
  2. bThe Rockefeller Graduate Program, The Rockefeller University, New York, NY 10065;
  3. cThe Tri-Institutional MD-PhD Program, The Rockefeller University, New York, NY 10065;
  4. dLaboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, NY 10065;
  5. eLaboratory of Molecular Neuro-Oncology, The Rockefeller University, New York, NY 10065;
  6. fDepartment of Neurobiology, Care Sciences and Society, Division of Neurogeriatrics, Center for Alzheimer Research, Karolinska Institutet, 141 57 Huddinge, Sweden;
  7. gThe Neuroimmunology and Inflammation Program, The Rockefeller University, New York, NY 10065;
  8. hDivision of Immune Diversity, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany;
  9. iHarold and Margaret Milliken Hatch Laboratory of Neuroendocrinology, The Rockefeller University, New York, NY 10065
  1. Contributed by Bruce S. McEwen, October 30, 2017 (sent for review August 14, 2017; reviewed by Silvo Conticello and Jin Billy Li)

  1. Fig. 2.

    APOBEC1 editing of a highly edited 3′UTR can modulate protein production. (A) Representative subclone sequencing of the 3′UTR of Cd36 genomic DNA (gDNA) and cDNA, derived from wildtype and Apobec1?/? BMDMs. (B) Relative luciferase levels of the Cd36 3′UTR with single editing events pre-encoded into DNA, in the absence of the editing enzyme (Cd36; n = 5). UN, unedited construct. (C) Mean fluorescence intensity (MFI) of CD36 surface protein levels in wildtype and Apobec1?/? BMDMs (n = 4). Error bars represent the SEM; statistical significance was obtained using a t test.

  2. Fig. 3.

    APOBEC1-mediated editing modulates protein production by altering 3′UTR regulation. (A) Effect of editing in protein production, in the absence of the editing enzyme. The 3′UTRs of interest depicted at the top of each plot were cloned directly from cDNA derived from wildtype BMDMs. A schematic at the top of each graph depicts the range of edited 3′UTRs tested. All error bars represent the SEM; statistical significance was obtained using a t test. LUC, luciferase. (B) Putative miRNA targets in APOBEC1-edited regions that overlap with Ago footprints. “Edited” (with C-to-T mutations reflecting APOBEC1-dependent changes) and “Unedited” (reflecting the genomic reference) footprint sequences were scanned for miRNA target regions (match to position 2–7, 1–6, or 3–8 of mature miRNA sequence). The miRNA targets that would be created (green) or disrupted (red) by an APOBEC1-editing event are depicted. (C and D) APOBEC1-editing disruption of putative miRNA target regions in the Sptssa and Rac1 3′UTRs. UN, unedited construct with a sequence consistent with the reference genome. ED, edited construct, mutated to reflect the editing event in question. A schematic depicting miRNA-site deletion or creation by APOBEC1 editing is shown at the top of each graph. The miRNA repression was calculated using the ratio of relative luciferase values (Materials and Methods) between miRNA and unrelated miRNA for each edited and unedited pair. The star indicates values below 0 or no relative repression (n = 5).

  3. Fig. 4.

    APOBEC1 is required for the proper phagocytosis and migration of BMDMs. (A) Phagocytosis of S. aureus pHrodo particles. (Left) Schematic of the phagocytosis setup: Phrodo-labeled particles are nonfluorescent in cell culture media; however, upon phagocytosis, they are transported to the lysosome inside the cell, whose acidic environment allows the particle to become fluorescent. (Right) Phagocytosis assay (n = 5). Error bars represent the SEM; statistical significance was obtained using a t test. MOI, multiplicity of infection. (B) Transendothelial migration assay. (Left) Schematic of the migration setup: Cells are plated in a two-chamber well (Top blue), which separates the cells from the chemokine (purple) via a porous membrane (green). Cells then transverse the membrane and can be quantified. (Right) Quantification of migration toward CXCL12. Error bars represent the SEM; statistical analysis was performed using the multiple measured one-way ANOVA, followed by a t test with Bonferroni’s correction (n = 3). *P < 0.05; **P < 0.01; ***P < 0.0001.

  4. Fig. 5.

    APOBEC1 is expressed within specific monocyte progenitors, and is required for the proper maintenance of monocyte populations in the periphery. (A) Schematic depicting the differentiation of monocytes from the cMOP. MDP, monocyte DC progenitor. (B) Apobec1 expression in sorted monocyte progenitors, relative to Gapdh expression, determined via quantitative PCR. (C) Schematic of the competitive reconstitution assay. Briefly, progenitor cells from both lineages were obtained from bone marrow and introduced into a recipient, whose progenitors were depleted via sublethal irradiation. The recipient mouse reconstitutes all blood lineages from the mixed wildtype and Apobec1?/? progenitors. (D) Analysis of bone marrow monocyte progenitor populations in radiation chimeras reconstituted with equal numbers of wildtype and Apobec1?/? progenitor cells 6 wk after transplantation (n = 6). (E) Analysis of spleens from mice reconstituted as in D. Error bars in D and E represent the SEM with statistical significance calculated using a t test.

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