| 1995 |
APOBEC1 (apobec-1) has distinct functional domains: zinc-coordinating residues (His61, Cys93, Cys96) and catalytic Glu63 are required for both cytidine deaminase activity and apoB RNA editing; the leucine-rich region (LRR) is required for RNA editing but not cytidine deaminase activity; His61 is required for RNA binding. Dominant negative mutants (Glu63→Gln, His61→Arg) reduce endogenous editing when overexpressed. |
Site-directed mutagenesis of GST-fusion APOBEC1, in vitro cytidine deaminase assay, apoB RNA editing assay, UV cross-linking RNA binding assay, transfection into McA 7777 cells |
The Journal of biological chemistry |
High |
7782343
|
| 1995 |
Recombinant APOBEC1 binds apoB RNA with high specificity via UV cross-linking and EMSA; binding is competed by poly(U) and poly(A,U) but not poly(A); RNA binding specificity for editing is distinct from mooring-sequence dependence. |
UV cross-linking, electrophoretic mobility shift assay (EMSA), RNA competition assays with recombinant GST/APOBEC-1 |
The Journal of biological chemistry |
High |
7782342
|
| 1996 |
Targeted disruption of mouse apobec-1 gene abolishes all apoB mRNA editing and eliminates apoB48 in serum, demonstrating that Apobec-1 is the essential catalytic component with no functional gene duplication. |
Gene targeting/knockout mice, RNA analysis of apoB mRNA editing, serum apoB48 Western blot |
The Journal of biological chemistry |
High |
8621694 8824235
|
| 1996 |
APOBEC-1 overexpression in stable hepatoma cell lines causes promiscuous editing of cytidines 5' of the mooring sequence in addition to the canonical site, and this hyperediting is mooring-sequence dependent and proportional to APOBEC-1 levels. |
Stable cell lines overexpressing APOBEC-1, apoB RNA editing assay, sequencing of editing products |
The Journal of biological chemistry |
Medium |
8621694
|
| 1996 |
APOBEC-1 requires auxiliary proteins (complementing activity) for apoB mRNA editing in vitro; recombinant APOBEC-1 immobilized on beads can deplete complementing activity from extracts and reconstitute editing, and the complementing protein (65 kDa) interacts directly with APOBEC-1 independently of apoB mRNA. The C-terminal 59 amino acids of APOBEC-1 are not required for this interaction. |
APOBEC-1 affinity chromatography, reconstitution in vitro editing assay, size exclusion chromatography, glycerol gradient sedimentation |
The Journal of biological chemistry |
High |
8910449
|
| 1997 |
ABBP-1, a novel RNA-binding protein identified by yeast two-hybrid using apobec-1 as bait, binds apobec-1 via its glycine-rich C-terminal domain, binds apoB mRNA around the editing site, and is required for apoB mRNA editing (immunodepletion abolishes editing; antisense knockdown reduces editing). |
Yeast two-hybrid, deletion mapping, UV cross-linking to apoB mRNA, immunodepletion from active extracts, antisense knockdown in HepG2 cells |
The Journal of biological chemistry |
Medium |
8999813
|
| 1997 |
A dominant negative APOBEC-1 mutant (mu1: H61K/C93S/C96S) retains dimerization capacity with wild-type APOBEC-1 and inhibits its editing activity; two other inactive mutants with poor dimerization fail to inhibit, demonstrating that active APOBEC-1 functions as a homodimer and dimerization is required for dominant negative inhibition. |
In vitro editing assay, immunoprecipitation dimerization assay, adenoviral in vivo delivery to mouse liver |
The Journal of biological chemistry |
High |
8999814
|
| 1998 |
The 65-kDa complementing activity (later identified as ACF) binds specifically to the mooring sequence of apoB mRNA (not antisense or mooring-sequence mutants) in the absence of apobec-1, and also interacts with apobec-1, supporting a model where it is the RNA-binding subunit of the holoenzyme. |
RNA affinity chromatography, UV cross-linking, far-Western analysis |
Molecular and cellular biology |
High |
9671452
|
| 2000 |
APOBEC1 complementation factor (ACF), a 64.3-kDa protein with three RNA recognition motifs, is identified and purified; ACF and apobec-1 constitute the minimal protein requirements for apoB mRNA editing in vitro. ACF binds apoB mRNA via the mooring sequence (UV cross-linking, immunoprecipitation), and ACF-apobec-1 complex forms in transfected cells. Immunodepletion of ACF from liver extracts abolishes editing. |
Protein purification, peptide sequencing, molecular cloning, UV cross-linking, immunoprecipitation, co-immunoprecipitation, immunodepletion, in vitro editing reconstitution |
Molecular and cellular biology |
High |
10669759
|
| 2000 |
APOBEC1 binds AU-rich sequences with consensus UUUN[A/U]U (Kd ~50 nM for high-affinity sites, ~435 nM for apoB RNA); binding of APOBEC1 to this consensus in c-myc 3'UTR stabilizes c-myc mRNA (half-life increased from 90 to 240 min), an effect abolished by RNA-binding–deficient APOBEC1 mutants. |
Filter binding assays, circular permutation analysis, RNA secondary structure prediction, mRNA half-life assay with actinomycin D, transfection of APOBEC1 expression mutants |
Molecular and cellular biology |
High |
10688645
|
| 2000 |
GRY-RBP, an RNA-binding protein with ~50% homology to ACF, is a component of the apoB mRNA editosome; it binds both ACF and apobec-1, binds apoB RNA, and inhibits C-to-U editing by sequestering ACF (competitive inhibition rescued by excess ACF). Antisense knockdown of GRY-RBP in hepatoma cells increases apoB RNA editing. |
Yeast two-hybrid, two-hybrid screen, peptide sequencing of active fraction, recombinant protein binding assays, immunodepletion, antisense knockdown, co-localization in transfected cells |
The Journal of biological chemistry |
High |
11134005
|
| 2001 |
CUGBP2 is a component of the apoB mRNA editing holoenzyme; it co-fractionates with ACF, immunodepletion of CUGBP2 co-precipitates ACF, CUGBP2 binds apoB RNA at an AU-rich sequence upstream of the edited cytidine, and addition of recombinant CUGBP2 dose-dependently inhibits C-to-U editing rescued by apobec-1 or ACF. |
Co-fractionation, immunodepletion, co-immunoprecipitation, UV cross-linking, in vitro editing reconstitution, antisense knockdown |
The Journal of biological chemistry |
High |
11577082
|
| 2001 |
ARCD-1, an apobec-1 homologue, has cytidine deaminase and apoB RNA binding activity but cannot mediate C-to-U RNA editing; it interacts with and heterodimerizes with apobec-1 and ACF, acting as a dominant negative inhibitor of apoB mRNA editing. |
Recombinant protein activity assays, heterodimerization immunoprecipitation, in vitro and in vivo editing assays, subcellular localization |
American journal of physiology. Cell physiology |
Medium |
11698249
|
| 2001 |
ABBP-2, a DnaJ/Hsp40 homologue identified by yeast two-hybrid, binds apobec-1 via its J domain and G/F domain; knockdown of ABBP-2 inhibits apoB mRNA editing; editing activity of tissue extracts requires Hsp70/ABBP-2 and is abolished by removal of endogenous ATP (which disrupts ABBP-2–Hsp70 interaction), implicating a chaperone function in editosome assembly. |
Yeast two-hybrid, domain deletion mapping, transfection GFP fusion, antisense knockdown in cells, in vitro editing assay with ATP depletion, Hsp70 co-IP |
The Journal of biological chemistry |
Medium |
11584023
|
| 2001 |
ACF domains required for RNA binding and apobec-1 interaction were mapped: the N-terminal RRMs (especially RRM1 and RRM2) together with the pre-RRM region are necessary for apoB mRNA binding and complementing activity; the auxiliary domain RG-rich region is required for RNA binding and apobec-1 interaction; the RRMs are also required for apobec-1 interaction. |
Deletion and point mutagenesis of ACF, in vitro binding assays (Kd measurements), in vitro editing complementation assay |
The Journal of biological chemistry |
High |
11571303
|
| 2001 |
ACF is a nuclear protein that upon co-transfection with apobec-1 promotes nuclear co-localization of apobec-1 (which is otherwise predominantly cytoplasmic); protein–protein interaction between ACF and apobec-1 is required for nuclear import of apobec-1; ACF NLS mutants that cannot interact with apobec-1 leave apobec-1 in the cytoplasm. |
Transfection of ACF/APOBEC1 constructs, immunofluorescence co-localization, deletion/mutation of NLS |
The Journal of biological chemistry |
Medium |
11571303
|
| 2002 |
ACF binds single-stranded but not double-stranded apoB mRNA with high affinity; all three RRMs contribute to RNA binding; point mutations in RRM1 or RRM2 decrease Kd for apoB mRNA by two orders of magnitude; the pre-RRM region is required for complementing activity and RNA binding but not for apobec-1 interaction. |
Deletion mutagenesis, point mutagenesis, binding assays with Kd measurements, in vitro editing complementation assay |
RNA |
High |
11871661
|
| 2002 |
APOBEC1 and its homologs APOBEC3C and APOBEC3G exhibit DNA mutator activity in E. coli through cytosine deamination (dC→dU), with each protein showing distinct local target sequence specificity. |
E. coli rifampicin-resistance mutation assay, expression of APOBEC1 and homologs in bacteria |
Molecular cell |
Medium |
12453430
|
| 2003 |
APOBEC1 deaminates cytosine to uracil in single-stranded DNA in vitro (using partially purified APOBEC1 from E. coli extracts); activity is specific for ssDNA (not dsDNA) and exhibits local sequence context dependence. |
In vitro biochemical deamination assay using partially purified recombinant APOBEC1, substrate specificity characterization |
The Journal of biological chemistry |
High |
12697753
|
| 2003 |
ACF localizes to the nucleus via a novel 41-residue nuclear localization signal (ANS) in its auxiliary domain; ACF is a nucleocytoplasmic shuttling protein (confirmed by heterokaryon assay); nuclear accumulation is CRM1-dependent for export and transportin-2–dependent for import; transportin 2 binds directly to the ANS motif. |
GFP/β-galactosidase chimera nuclear localization assay, actinomycin D treatment, leptomycin B CRM1 inhibition, heterokaryon assay, co-immunoprecipitation with transportin 2, confocal microscopy |
The Journal of biological chemistry |
Medium |
12896982
|
| 2004 |
Crystal structure of yeast CDD1, an APOBEC1 ortholog, at 2.0-Å resolution reveals the fold; comparative modeling of APOBEC1 indicates dimerization and trans-acting loops contributing to active site formation; a central active site 'flap' accommodates large substrates (RNA or ssDNA), explaining equal likelihood of APOBEC1 binding ssDNA or RNA. |
X-ray crystallography of CDD1, comparative structural modeling of APOBEC1 and AID |
Proceedings of the National Academy of Sciences of the United States of America |
Medium |
15148397
|
| 2004 |
Purified APOBEC1 apoenzyme has intrinsic residual editing activity on a minimal apoB mRNA substrate without auxiliary factors; ACF broadens the temperature range and lowers the optimal temperature for editing activity, consistent with ACF promoting a conformational transition in the RNA substrate. |
Expression and purification to homogeneity of APOBEC1, in vitro editing kinetics assay with and without ACF, factorial and response surface experimental design |
RNA |
High |
15273326
|
| 2004 |
Apobec-1 binds to AU-rich sequences in the COX-2 mRNA 3'UTR (first 60 nt) via EMSA and UV cross-linking, stabilizing COX-2 mRNA; apobec-1-knockout mice show reduced COX-2 induction and intestinal stem cell survival after gamma-irradiation, an effect mediated through COX-2/prostaglandin E2. |
EMSA, UV cross-linking, mRNA half-life assay with chimeric luciferase-COX-2 3'UTR reporter, apobec-1 knockout mice with irradiation model, real-time PCR, Western blot |
Gastroenterology |
High |
15480992
|
| 2005 |
NMR structure of the 31-nt apoB mRNA stem-loop shows the edited cytidine (C6666) is stacked in a loop and inaccessible to APOBEC1 alone; APOBEC1 does not specifically bind apoB mRNA by itself but requires ACF, which recognizes the flexible mooring sequence and melts the stem-loop to expose C6666 to APOBEC1. |
NMR structure determination of apoB mRNA stem-loop, RNA binding assays with APOBEC1 and ACF |
RNA |
High |
15659357
|
| 2006 |
APOBEC1 and AID are nucleo-cytoplasmic trafficking proteins, whereas APOBEC3G is strongly retained in the cytoplasm through mechanisms involving both N- and C-terminal regions. |
Subcellular fractionation, nucleo-cytoplasmic shuttling assays in cells |
Biochemical and biophysical research communications |
Medium |
16999936
|
| 2006 |
ACF phosphorylation (on serine residues, predominantly by protein phosphatase I-sensitive kinase) regulates apoB mRNA editing: phosphorylated ACF is restricted to nuclei and co-sediments with editing-competent 27S complexes; alkaline phosphatase treatment reduces APOBEC-1 co-immunoprecipitation with ACF and inhibits editing; ethanol stimulates both ACF phosphorylation and editing. |
Co-immunoprecipitation, alkaline phosphatase treatment of nuclear extracts, glycerol gradient sedimentation, two-dimensional phosphoamino acid analysis, protein phosphatase inhibitor treatment in primary hepatocytes |
Nucleic acids research |
High |
16820530
|
| 2006 |
ACF phosphorylation at S154 and S368 by PKC regulates apoB mRNA editing; PKC activation stimulates editing and ACF phosphorylation; S154A/S368A mutations inhibit ethanol-stimulated editing; S154D/S368D phosphomimetics stimulate editing to levels comparable to ethanol treatment. |
PKC/PKA pharmacological activation in primary hepatocytes, in vitro phosphorylation of purified ACF by PKC, site-directed mutagenesis (alanine/aspartate substitutions), in vitro editing assay |
Biochimica et biophysica acta |
High |
17229474
|
| 2007 |
Deletion of apobec-1 in Apc(min/+) mice dramatically reduces intestinal adenoma formation, associated with increased apoptosis, reduced proliferation, reduced COX-2 mRNA (~2-fold vs. adenomas), and reduced expression of other AU-rich mRNA targets (EGFR, PPARδ, EP4, c-myc) containing the apobec-1 consensus binding site. Adenoviral apobec-1 in HCA-7 cells dose-dependently increases COX-2 protein and stabilizes COX-2 mRNA. |
Genetic epistasis (compound Apc(min/+) apobec-1(-/-) mice), intestinal adenoma counting, apoptosis and proliferation assays, mRNA quantification, adenoviral apobec-1 delivery in cancer cells |
Cancer research |
High |
17875695
|
| 2009 |
APOBEC1 binds to AU-rich regions of the Cyp7a1 mRNA 3'UTR containing the UUUN(A/U)U consensus, as shown by UV cross-linking and in vivo RNA co-immunoprecipitation; Apobec-1-knockout mice show decreased Cyp7a1 mRNA and protein (without changes in transcription), increased gallstone susceptibility, and adenoviral Apobec-1 rescue restores Cyp7a1 expression. |
UV cross-linking to recombinant APOBEC1, in vivo RNA co-immunoprecipitation, Cyp7a1 transcription run-on, adenoviral rescue in knockout mice, lithogenic diet model |
The Journal of biological chemistry |
High |
19386592
|
| 2011 |
APOBEC1 edits multiple mRNA targets (32 validated) located in AU-rich segments of transcript 3'UTRs, identified by transcriptome-wide comparative RNA-Seq of APOBEC1-expressing vs. non-expressing conditions; editing sites share characteristic sequence features. |
Transcriptome-wide comparative RNA-Seq (APOBEC1+ vs. control), Sanger sequence validation of editing sites |
Nature structural & molecular biology |
High |
21258325
|
| 2011 |
APOBEC1 (A1) restricts LINE-1 and LTR retrotransposons (IAP, MusD) in cell-culture retrotransposition assays; anti-L1 activity is deamination-independent, whereas inhibition of LTR-retrotransposons requires deaminase activity; restriction is not affected by subcellular localization. |
Cell culture-based retrotransposition assays, deaminase-inactive mutant controls, subcellular localization experiments |
Nucleic acids research |
Medium |
21398638
|
| 2014 |
RBM47 is a novel RNA-binding protein that interacts with APOBEC1 and A1CF; RBM47 can substitute for A1CF in APOBEC1-mediated C-to-U RNA editing in vitro and is necessary and sufficient for APOBEC1-mediated editing; Rbm47-deficient mice exhibit impaired RNA editing. |
Co-immunoprecipitation of RBM47 with APOBEC1 and A1CF, in vitro editing assay with RBM47, Rbm47 knockout mice |
EMBO reports |
High |
24916387
|
| 2014 |
Genome-wide deep sequencing of intestinal and hepatic RNA from wild-type and Apobec-1-deficient mice reveals 56 novel editing sites in 54 intestinal mRNAs and 22 sites in 17 liver mRNAs (all in 3'UTRs); editing leads to corresponding changes in intestinal mRNA and protein levels for 11 genes; Apobec-1 adenoviral or transgenic rescue restores editing; cell-free extracts from wild-type but not Apobec-1(-/-) mice support editing. |
Deep sequencing (RNA-Seq), Sanger-sequence validation, tissue-specific Apobec-1 adenoviral and transgenic overexpression rescue, cell-free editing assays, polysome profiling |
Genome biology |
High |
24946870
|
| 2017 |
A1CF (APOBEC1 complementation factor) is dispensable for C-to-U RNA editing in vivo under normal physiological conditions: A1cf conditional null mice are viable and fertile, with no changes in editing efficiency at multiple targets including ApoB in small intestine and liver. |
Conditional A1cf knockout mice, quantitative C-to-U RNA editing at multiple targets in small intestine and liver |
RNA |
High |
28069890 30309881
|
| 2017 |
APOBEC1-mediated RNA editing occurs in microglia and is required for maintaining their resting state; mice lacking APOBEC1 editing function in microglia display progressive age-related neurodegeneration, activated microglial clustering, aberrant myelination, increased inflammation, and lysosomal anomalies with behavioral and motor deficiencies. |
APOBEC1 RNA editing analysis in microglia, conditional APOBEC1 knockout mice, histopathological and behavioral phenotyping |
Proceedings of the National Academy of Sciences of the United States of America |
Medium |
29167375
|
| 2018 |
A1CF and RBM47 each function independently but interact in a tissue-specific manner to regulate APOBEC1-dependent C-to-U RNA editing; double knockout of A1cf and Rbm47 in liver virtually eliminates apoB RNA editing and reduces most other targets; intestinal double knockout further reduces editing beyond single Rbm47 knockout; forced A1CF expression alone does not restore editing in double knockouts. |
Tissue-specific single and double conditional knockouts of A1cf and Rbm47, adenoviral APOBEC1 rescue, RNA-Seq, quantitative editing analysis |
RNA |
High |
30309881
|
| 2019 |
APOBEC1-RBM47 and APOBEC1-A1CF complexes reconstituted in HEK293T cells show differential RNA editing activity: A1CF and RBM47 differ in their selectivity for specific RNA targets; human vs. mouse versions of cofactors differ in activity; minimal domain of RBM47 required for activity was defined. |
Reconstitution of APOBEC1 with either cofactor in HEK293T cells, cell-based fluorescent editing reporter assay, domain deletion analysis of RBM47 |
Journal of molecular biology |
Medium |
30844405
|
| 2020 |
Crystal structure of APOBEC1 reveals a typical APOBEC deaminase core plus a unique well-folded hydrophobic C-terminal domain (A1HD) that forms a stable dimer via hydrophobic interactions creating a four-stranded β-sheet positively charged surface; structure-guided mutagenesis shows A1HD directs RNA substrate and cofactor interactions. |
X-ray crystallography of APOBEC1, structure-guided mutagenesis, biochemical characterization of RNA/DNA substrate selectivity |
NAR cancer |
High |
33094286
|
| 2021 |
APOBEC1 cytosine deaminase activity on ssDNA is suppressed by replication protein A (RPA) competition for ssDNA; APOBEC1 cannot efficiently compete with RPA in vitro, correlating with low levels of genomic DNA damage (γH2AX foci) in lung cancer cells, suggesting RPA acts as a defense against APOBEC1 off-target deamination. |
In vitro ssDNA deamination assay with RPA competition, γH2AX foci assay in lung cancer cell line |
Nucleic acids research |
Medium |
33330905
|
| 1999 |
APOBEC1 dimerization requires the C-terminal region; N-terminal deletions up to residue A117 do not impair dimerization, while C-terminal deletions reduce it; basic amino acid clusters R15R16R17 and R33K34 are essential for apoB mRNA editing; residues L182, I185, L189 and a β-turn (P190/P191) in the leucine-rich C-terminal region are required for normal editing function. |
Systematic targeted mutagenesis, in vitro apoB mRNA editing assay, immunoprecipitation dimerization assay |
Journal of lipid research |
High |
10191286
|
| 2000 |
In yeast expressing APOBEC-1, apoB mRNA editing occurs preferentially in the nucleus; cis-acting sequence requirements and intracellular distribution of APOBEC-1 in yeast are similar to those in mammalian cells, suggesting auxiliary protein functions required for editosome assembly are conserved in yeast. |
Yeast expression system for APOBEC-1, apoB mRNA editing assay, intracellular localization |
Nucleic acids research |
Medium |
10606639
|
| 2014 |
APOBEC1 expression in chicken DT40 cells increases inactivation of a stably inserted reporter gene and increases imatinib-resistant clones in a human CML model through induction of mutations in BCR-ABL1, demonstrating APOBEC1 acts as a DNA mutator in vertebrate cells. |
Reporter gene inactivation assay in DT40 cells, imatinib-resistance mutation induction assay in human CML cells, BCR-ABL1 sequencing |
Genome biology |
Medium |
25085003
|
| 2014 |
hnRNPQ isoform 6 (hnRNPQ6) is required for efficient interaction of APOBEC1 with IL-8 mRNA in HuH7.5 cells; APOBEC1 binding to AU-rich elements in IL-8 3'UTR extends mRNA half-life and increases IL-8 production in a cell-type-specific and hnRNPQ6-dependent manner. |
FLAG-tagged APOBEC1 immunoprecipitation followed by microarray (RNA-IP), siRNA screen, yeast two-hybrid, reporter assay with IL-8 3'UTR-luciferase, mRNA half-life measurement, overexpression of hnRNPQ isoforms |
The Journal of biological chemistry |
Medium |
25100733
|