APOBEC3G Restricts HIV-1 to a Greater Extent than APOBEC3F and APOBEC3DE in Human Primary CD4+ T Cells and Macrophages

APOBEC3 proteins inhibit HIV-1 replication in experimental systems and induce hypermutation in infected patients; however, the relative contributions of several APOBEC3 proteins to restriction of HIV-1 replication in the absence of the viral Vif protein in human primary CD4⁺ T cells and macrophages are unknown. We observed significant inhibition of HIV-1Δvif produced in 293T cells in the presence of APOBEC3DE (A3DE), APOBEC3F (A3F), APOBEC3G (A3G), and APOBEC3H haplotype II (A3H HapII) but not APOBEC3B (A3B), APOBEC3C (A3C), or APOBEC3H haplotype I (A3H HapI). Our previous studies showed that Vif amino acids Y⁴⁰RHHY⁴⁴ are important for inducing proteasomal degradation of A3G, whereas amino acids ¹⁴DRMR¹⁷ are important for degradation of A3F and A3DE. Here, we introduced substitution mutations of ⁴⁰YRHHY⁴⁴ and ¹⁴DRMR¹⁷ in replication-competent HIV-1 to generate vif mutants NL4-3 YRHHY>A5 and NL4-3 DRMR>A4 to compare the antiviral activity of A3G to the combined antiviral activity of A3F and A3DE in activated CD4⁺ T cells and macrophages. During the first 15 days (round 1), in which multiple cycles of viral replication occurred, both the NL4-3 YRHHY>A5 and NL4-3 DRMR>A4 mutants replicated in activated CD4⁺ T cells and macrophages, and only the NL4-3 YRHHY>A5 mutant showed a 2- to 4-day delay in replication compared to the wild type. During the subsequent 27 days (round 2) of cultures initiated with peak virus obtained from round 1, the NL4-3 YRHHY>A5 mutant exhibited a longer, 8- to 10-day delay and the NL4-3 DRMR>A4 mutant exhibited a 2- to 6-day delay in replication compared to the wild type. The NL4-3 YRHHY>A5 and NL4-3 DRMR>A4 mutant proviruses displayed G-to-A hypermutations primarily in GG and GA dinucleotides as expected of A3G- and A3F- or A3DE-mediated deamination, respectively. We conclude that A3G exerts a greater restriction effect on HIV-1 than A3F and A3DE.     

Tumor Necrosis Factor (TNF) –308G>A,Nitric Oxide Synthase 3 (NOS3) +894G>T Polymorphisms and Migraine Risk: A Meta-Analysis

Background and Objective

Conflicting data have been reported on the association between tumor necrosis factor (TNF) –308G>A and nitric oxide synthase 3 (NOS3) +894G>T polymorphisms and migraine. We performed a meta-analysis of case-control studies to evaluate whether the TNF –308G>A and NOS3 +894G>T polymorphisms confer genetic susceptibility to migraine.

Method

We performed an updated meta-analysis for TNF –308G>A and a meta-analysis for NOS3 +894G>T based on studies published up to July 2014. We calculated study specific odds ratios (OR) and 95% confidence intervals (95% CI) assuming allele contrast, dominant model, recessive model, and co-dominant model as pooled effect estimates.

Results

Eleven studies in 6682 migraineurs and 22591 controls for TNF –308G>A and six studies in 1055 migraineurs and 877 controls for NOS3 +894G>T were included in the analysis. Neither indicated overall associations between gene polymorphisms and migraine risk. Subgroup analyses suggested that the “A” allele of the TNF –308G>A variant increases the risk of migraine among non-Caucasians (dominant model: pooled OR = 1.82; 95% CI 1.15 – 2.87). The risk of migraine with aura (MA) was increased among both Caucasians and non-Caucasians. Subgroup analyses suggested that the “T” allele of the NOS3 +894G>T variant increases the risk of migraine among non-Caucasians (co-dominant model: pooled OR = 2.10; 95% CI 1.14 – 3.88).

Conclusions

Our findings appear to support the hypothesis that the TNF –308G>A polymorphism may act as a genetic susceptibility factor for migraine among non-Caucasians and that the NOS3 +894G>T polymorphism may modulate the risk of migraine among non-Caucasians.     

FANCA Gene Mutations with 8 Novel Molecular Changes in Indian Fanconi Anemia Patients

Fanconi anemia (FA), a rare heterogeneous genetic disorder, is known to be associated with 19 genes and a spectrum of clinical features. We studied FANCA molecular changes in 34 unrelated and 2 siblings of Indian patients with FA and have identified 26 different molecular changes of FANCA gene, of which 8 were novel mutations (a small deletion c.2500delC, 4 non-sense mutations c.2182C>T, c.2630C>G, c.3677C>G, c.3189G>A; and 3 missense mutations; c.1273G>C, c.3679 G>C, and c.3992 T>C). Among these only 16 patients could be assigned FA-A complementation group, because we could not confirm single exon deletions detected by MLPA or cDNA amplification by secondary confirmation method and due to presence of heterozygous non-pathogenic variations or heterozygous pathogenic mutations. An effective molecular screening strategy should be developed for confirmation of these mutations and determining the breakpoints for single exon deletions.     

DEFB1 polymorphisms are involved in susceptibility to human papillomavirus infection in Brazilian gynaecological patients

The human beta defensin 1 (hBD-1) antimicrobial peptide is a member of the innate immune system known to act in the first line of defence against microorganisms, including viruses such as human papillomavirus (HPV). In this study, five functional polymorphisms (namely g-52G>A, g-44C>G and g-20G>A in the 5’UTR and c.*5G>A and c.*87A>G in the 3’UTR) in the DEFB1 gene encoding for hBD-1 were analysed to investigate the possible involvement of these genetic variants in susceptibility to HPV infection and in the development of HPV-associated lesions in a population of Brazilian women. The DEFB1 g-52G>A and c.*5G>A single-nucleotide polymorphisms (SNPs) and the GCAAA haplotype showed associations with HPV-negative status; in particular, the c.*5G>A SNP was significantly associated after multiple test corrections. These findings suggest a possible role for the constitutively expressed beta defensin-1 peptide as a natural defence against HPV in the genital tract mucosa.     

Footprint of APOBEC3 on the Genome of Human Retroelements

Almost half of the human genome is composed of transposable elements. The genomic structures and life cycles of some of these elements suggest they are a result of waves of retroviral infection and transposition over millions of years. The reduction of retrotransposition activity in primates compared to that in nonprimates, such as mice, has been attributed to the positive selection of several antiretroviral factors, such as apolipoprotein B mRNA editing enzymes. Among these, APOBEC3G is known to mutate G to A within the context of GG in the genome of endogenous as well as several exogenous retroelements (the underlining marks the G that is mutated). On the other hand, APOBEC3F and to a lesser extent other APOBEC3 members induce G-to-A changes within the nucleotide GA. It is known that these enzymes can induce deleterious mutations in the genome of retroviral sequences, but the evolution and/or inactivation of retroelements as a result of mutation by these proteins is not clear. Here, we analyze the mutation signatures of these proteins on large populations of long interspersed nuclear element (LINE), short interspersed nuclear element (SINE), and endogenous retrovirus (ERV) families in the human genome to infer possible evolutionary pressure and/or hypermutation events. Sequence context dependency of mutation by APOBEC3 allows investigation of the changes in the genome of retroelements by inspecting the depletion of G and enrichment of A within the APOBEC3 target and product motifs, respectively. Analysis of approximately 22,000 LINE-1 (L1), 24,000 SINE Alu, and 3,000 ERV sequences showed a footprint of GG→AG mutation by APOBEC3G and GA→AA mutation by other members of the APOBEC3 family (e.g., APOBEC3F) on the genome of ERV-K and ERV-1 elements but not on those of ERV-L, LINE, or SINE.     

Characterisation of Gut Microbiota in Ossabaw and G?ttingen Minipigs as Models of Obesity and Metabolic Syndrome

Background

Recent evidence suggests that the gut microbiota is an important contributing factor to obesity and obesity related metabolic disorders, known as the metabolic syndrome. The aim of this study was to characterise the intestinal microbiota in two pig models of obesity namely Göttingen minipigs and the Ossabaw minipigs.

Methods and Findings

The cecal, ileal and colonic microbiota from lean and obese Osabaw and Göttingen minipigs were investigated by Illumina-based sequencing and by high throughput qPCR, targeting the 16S rRNA gene in different phylogenetic groups of bacteria. The weight gain through the study was significant in obese Göttingen and Ossabaw minipigs. The lean Göttingen minipigs’ cecal microbiota contained significantly higher abundance of Firmicutes (P<0.006), Akkermensia (P<0.01) and Methanovibribacter (P<0.01) than obese Göttingen minipigs. The obese Göttingen cecum had higher abundances of the phyla Spirochaetes (P<0.03), Tenericutes (P<0.004), Verrucomicrobia (P<0.005) and the genus Bacteroides (P<0.001) compared to lean minipigs. The relative proportion of Clostridium cluster XIV was 7.6-fold higher in cecal microbiota of obese Göttingen minipigs as compared to lean. Obese Ossabaw minipigs had a higher abundance of Firmicutes in terminal ileum and lower abundance of Bacteroidetes in colon than lean Ossabaw minipigs (P<0.01). Obese Ossabaws had significantly lower abundances of the genera Prevotella and Lactobacillus and higher abundance of Clostridium in their colon than the lean Ossabaws. Overall, the Göttingen and Ossabaw minipigs displayed different microbial communities in response to diet-induced obesity in the different sections of their intestine.

Conclusion

Obesity-related changes in the composition of the gut microbiota were found in lean versus obese Göttingen and Ossabaw minipigs. In both pig models diet seems to be the defining factor that shapes the gut microbiota as observed by changes in different bacteria divisions between lean and obese minipigs.     

Human APOBEC3 Induced Mutation of Human Immunodeficiency Virus Type-1 Contributes to Adaptation and Evolution in Natural Infection

Human APOBEC3 proteins are cytidine deaminases that contribute broadly to innate immunity through the control of exogenous retrovirus replication and endogenous retroelement retrotransposition. As an intrinsic antiretroviral defense mechanism, APOBEC3 proteins induce extensive guanosine-to-adenosine (G-to-A) mutagenesis and inhibit synthesis of nascent human immunodeficiency virus-type 1 (HIV-1) cDNA. Human APOBEC3 proteins have additionally been proposed to induce infrequent, potentially non-lethal G-to-A mutations that make subtle contributions to sequence diversification of the viral genome and adaptation though acquisition of beneficial mutations. Using single-cycle HIV-1 infections in culture and highly parallel DNA sequencing, we defined trinucleotide contexts of the edited sites for APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H. We then compared these APOBEC3 editing contexts with the patterns of G-to-A mutations in HIV-1 DNA in cells obtained sequentially from ten patients with primary HIV-1 infection. Viral substitutions were highest in the preferred trinucleotide contexts of the edited sites for the APOBEC3 deaminases. Consistent with the effects of immune selection, amino acid changes accumulated at the APOBEC3 editing contexts located within human leukocyte antigen (HLA)-appropriate epitopes that are known or predicted to enable peptide binding. Thus, APOBEC3 activity may induce mutations that influence the genetic diversity and adaptation of the HIV-1 population in natural infection.     

Recurrent Muscle Weakness with Rhabdomyolysis,Metabolic Crises,and Cardiac Arrhythmia Due to Bi-allelic TANGO2 Mutations

The underlying genetic etiology of rhabdomyolysis remains elusive in a significant fraction of individuals presenting with recurrent metabolic crises and muscle weakness. Using exome sequencing, we identified bi-allelic mutations in TANGO2 encoding transport and Golgi organization 2 homolog (Drosophila) in 12 subjects with episodic rhabdomyolysis, hypoglycemia, hyperammonemia, and susceptibility to life-threatening cardiac tachyarrhythmias. A recurrent homozygous c.460G>A (p.Gly154Arg) mutation was found in four unrelated individuals of Hispanic/Latino origin, and a homozygous ∼34 kb deletion affecting exons 3–9 was observed in two families of European ancestry. One individual of mixed Hispanic/European descent was found to be compound heterozygous for c.460G>A (p.Gly154Arg) and the deletion of exons 3–9. Additionally, a homozygous exons 4–6 deletion was identified in a consanguineous Middle Eastern Arab family. No homozygotes have been reported for these changes in control databases. Fibroblasts derived from a subject with the recurrent c.460G>A (p.Gly154Arg) mutation showed evidence of increased endoplasmic reticulum stress and a reduction in Golgi volume density in comparison to control. Our results show that the c.460G>A (p.Gly154Arg) mutation and the exons 3–9 heterozygous deletion in TANGO2 are recurrent pathogenic alleles present in the Latino/Hispanic and European populations, respectively, causing considerable morbidity in the homozygotes in these populations.     

The Frequency and Clinical Significance of IDH1 Mutations in Chinese Acute Myeloid Leukemia Patients

Objective

Mutations in the gene encoding isocitrate dehydrogenease 1 (IDH1) occur in various hematopoietic tumors including acute myeloid leukemia (AML), myeloproliferative neoplasms and myelodysplastic syndromes. IDH1 mutations are significant in both diagnosis and prognosis of these conditions. In the present study we determined the prevalence and clinical significance of IDH1 mutations in 349 samples from newly diagnosed AML patients.

Results

Of the 349 AML patient specimens analyzed, 35 (10.03%) were found to have IDH1 mutations including 4 IDH1 R132 mutations and 31 non-R132 mutations. IDH1 non-R132 mutations were largely concentrated within AML-M₁ (35.72%, p<0.01). We identified five IDH1 mutations that were novel to AML: (1) c.299 G>A, p.R100Q; (2) c.311G>T, p.G104V; (3) c.322T>C, p.F108L; (4) c.356G>A, p.R119Q; and (5) c.388A>G, p.I130V. In addition, we identified three IDH1 mutations that were previously described in AML. The frequency of IDH1 mutations in AML patients with normal karyotype was 9.9%. IDH1 non-R132 mutations were concurrent with mutations in FLT3-ITD (p<0.01), CEBPA (p<0.01), and NRAS (p<0.01), as well as the overexpression of MN1 (p<0.01) and WT1(p<0.01). The overall survival (OS) in the patients with IDH1 non-R132 mutations compared to patients without IDH1 mutations don''t reach statistically significance (median 521 days vs median: not reached; n.s.).

Conclusion

IDH1 non-R132 mutations occurred frequently in newly diagnosed adult Chinese AML patients, and these mutations were associated with genetic alterations. The OS was not influenced by IDH1 non-R132 mutations in the present study.     

Induction of Body Weight Loss through RNAi-Knockdown of APOBEC1 Gene Expression in Transgenic Rabbits

In the search of new strategies to fight against obesity, we targeted a gene pathway involved in energy uptake. We have thus investigated the APOB mRNA editing protein (APOBEC1) gene pathway that is involved in fat absorption in the intestine. The APOB gene encodes two proteins, APOB100 and APOB48, via the editing of a single nucleotide in the APOB mRNA by the APOBEC1 enzyme. The APOB48 protein is mandatory for the synthesis of chylomicrons by intestinal cells to transport dietary lipids and cholesterol. We produced transgenic rabbits expressing permanently and ubiquitously a small hairpin RNA targeting the rabbit APOBEC1 mRNA. These rabbits exhibited a moderately but significantly reduced level of APOBEC1 gene expression in the intestine, a reduced level of editing of the APOB mRNA, a reduced level of synthesis of chylomicrons after a food challenge, a reduced total mass of body lipids and finally presented a sustained lean phenotype without any obvious physiological disorder. Interestingly, no compensatory mechanism opposed to the phenotype. These lean transgenic rabbits were crossed with transgenic rabbits expressing in the intestine the human APOBEC1 gene. Double transgenic animals did not present any lean phenotype, thus proving that the intestinal expression of the human APOBEC1 transgene was able to counterbalance the reduction of the rabbit APOBEC1 gene expression. Thus, a moderate reduction of the APOBEC1 dependent editing induces a lean phenotype at least in the rabbit species. This suggests that the APOBEC1 gene might be a novel target for obesity treatment.     

Association of Mitochondrial DNA Polymerase γ Gene POLG1 Polymorphisms with Parkinsonism in Chinese Populations

Background

Mitochondrial DNA polymerase gamma (POLG1) mutations were associated with levodopa-responsive Parkinsonism. POLG1 gene contains a number of common nonsynonymous SNPs and intronic regulatory SNPs which may have functional consequences. It is of great interest to discover polymorphisms variants associated with Parkinson''s disease (PD), both in isolation and in combination with specific SNPs.

Materials and Methods

We conducted a case-control study and genotyped twenty SNPs and poly-Q polymorphisms of POLG1 gene including in 344 Chinese sporadic PD patients and 154 healthy controls. All the polymorphisms of POLG1 we found in this study were sequenced by PCR products with dye terminator methods using an ABI-3100 sequencer. Hardy-Weinberg equilibrium and linkage disequilibrium (LD) for association between twenty POLG1 SNPs and PD were calculated using the program Haploview.

Principal Results

We provided evidence for strong association of four intronic SNPs of the POLG1 gene (new report: c.2070-12T>A and rs2307439: c.2070-64G>A in intron 11, P = 0.00011, OR = 1.727; rs2302084: c.3105-11T>C and rs2246900: c.3105-36A>G in intron 19, P = 0.00031, OR = 1.648) with PD. However, we did not identify any significant association between ten exonic SNPs of POLG1 and PD. Linkage disequilibrium analysis indicated that c.2070-12T>A and c.2070-64G>A could be parsed into one block as Haplotype 1 as well as c.3105-11T>C and c.3105-36A>G in Haplotype 2. In addition, case and control study on association of POLG1 CAG repeat (poly-Q) alleles with PD showed a significant association (P = 0.03, OR = 2.16) of the non-10/11Q variants with PD. Although intronic SNPs associated with PD didn''t influence POLG1 mRNA alternative splicing, there was a strong association of c.2070-12T>A and c.2070-64G>A with decreased POLG1 mRNA level and protein levels.

Conclusions

Our findings indicate that POLG1 may play a role in the pathogenesis of PD in Chinese populations.     

APOBEC2 negatively regulates myoblast differentiation in muscle regeneration

Recently we found that the deficiency of APOBEC2, a member of apoB mRNA editing enzyme, catalytic polypeptide-like family, leads to a diminished muscle mass and increased myofiber with centrally-located nuclei known as dystrophic phenotypes. APOBEC2 expression is predominant in skeletal and cardiac muscles and elevated exclusively at the early-differentiation phase of wild-type (WT) myoblast cultures; however the physiological significance is still un-known. Here we show that APOBEC2 is a key negative regulator of myoblast differentiation in muscle regeneration. APOBEC2-knockout (A2KO) mice myoblast cultures displayed a normal morphology of primary myotubes along with earlier increase in fusion index and higher expression levels of myosin heavy chain (MyHC), myogenin and its cooperating factor MEF2C than WT myoblasts. Similar response was observable in APOBEC2-knockdown cultures of WT myoblasts that were transfected with the specific siRNA at the differentiation phase (not proliferation phase). Importantly, cardiotoxin-injured A2KO gastrocnemius muscle provided in vivo evidence by showing larger up-regulation of neonatal MyHC and myogenin and hence earlier regeneration of myofiber structures with diminished cross-sectional areas and minimal Feret diameters. Therefore, the findings highlight a promising role for APOBEC2 in normal progression of regenerative myogenesis at the early-differentiation phase upon muscle injury.     

Variants of the PPARD Gene and Their Clinicopathological Significance in Colorectal Cancer

Background

Peroxisome proliferator-activated receptor delta (PPARD) is nuclear hormone receptor involved in colorectal cancer (CRC) differentiation and progression. The purpose of this study was to determine prevalence and spectrum of variants in the PPARD gene in CRC, and their contribution to clinicopathological endpoints.

Methods and Findings

Direct sequencing of the PPARD gene was performed in 303 primary tumors, in blood samples from 50 patients with ≥3 affected first-degree relatives, 50 patients with 2 affected first-degree relatives, 50 sporadic patients, 360 healthy controls, and in 6 colon cancer cell lines. Mutation analysis revealed 22 different transversions, 7 of them were novel. Three of all variants were somatic (c.548A>G, p.Y183C, c.425-9C>T, and c.628-16G>A). Two missense mutations (p.Y183C and p.R258Q) were pathogenic using in silico predictive program. Five recurrent variants were detected in/adjacent to the exons 4 (c.1-87T>C, c.1-67G>A, c.130+3G>A, and c.1-101-8C>T) and exon 7 (c.489T>C). Variant c.489C/C detected in tumors was correlated to worse differentiation (P = 0.0397).

Conclusions

We found 7 novel variants among 22 inherited or acquired PPARD variants. Somatic and/or missense variants detected in CRC patients are rare but indicate the clinical importance of the PPARD gene.     

Human Tribbles 3 Protects Nuclear DNA from Cytidine Deamination by APOBEC3A

The human polydeoxynucleotide cytidine deaminases APOBEC3A, APOBEC3C, and APOBEC3H are capable of mutating viral DNA in the nucleus, whereas APOBEC3A alone efficiently edits nuclear DNA. Deamination is rapidly followed by excision of uracil residues and can lead to double-stranded breaks. It is not known to which protein networks these DNA mutators belong. Using a yeast two-hybrid screen, we identified the human homolog of Drosophila Tribbles 3, TRIB3, as an interactor for APOBEC3A and APOBEC3C. The interaction was confirmed by co-affinity purification. Co-transfection of APOBEC3A with a TRIB3 expression vector reduced nuclear DNA editing whereas siRNA knockdown of TRIB3 increased the levels of nuclear DNA editing, indicating that TRIB3 functioned as a repressor of A3A. It also repressed A3A-associated γH2AX positive double-stranded breaks. The interaction results in degradation of A3A in a proteasome-independent manner. TRIB3 has been linked to cancer and via its own interactors and links the A3A DNA mutators to the Rb-BRCA1-ATM network. TRIB3 emerges as an important guardian of genome integrity.     

Mutation Screening of Retinal Dystrophy Patients by Targeted Capture from Tagged Pooled DNAs and Next Generation Sequencing

Purpose

Retinal dystrophies are genetically heterogeneous, resulting from mutations in over 200 genes. Prior to the development of massively parallel sequencing, comprehensive genetic screening was unobtainable for most patients. Identifying the causative genetic mutation facilitates genetic counselling, carrier testing and prenatal/pre-implantation diagnosis, and often leads to a clearer prognosis. In addition, in a proportion of cases, when the mutation is known treatment can be optimised and patients are eligible for enrolment into clinical trials for gene-specific therapies.

Methods

Patient genomic DNA was sheared, tagged and pooled in batches of four samples, prior to targeted capture and next generation sequencing. The enrichment reagent was designed against genes listed on the RetNet database (July 2010). Sequence data were aligned to the human genome and variants were filtered to identify potential pathogenic mutations. These were confirmed by Sanger sequencing.

Results

Molecular analysis of 20 DNAs from retinal dystrophy patients identified likely pathogenic mutations in 12 cases, many of them known and/or confirmed by segregation. These included previously described mutations in ABCA4 (c.6088C>T,p.R2030*; c.5882G>A,p.G1961E), BBS2 (c.1895G>C,p.R632P), GUCY2D (c.2512C>T,p.R838C), PROM1 (c.1117C>T,p.R373C), RDH12 (c.601T>C,p.C201R; c.506G>A,p.R169Q), RPGRIP1 (c.3565C>T,p.R1189*) and SPATA7 (c.253C>T,p.R85*) and new mutations in ABCA4 (c.3328+1G>C), CRB1 (c.2832_2842+23del), RP2 (c.884-1G>T) and USH2A (c.12874A>G,p.N4292D).

Conclusions

Tagging and pooling DNA prior to targeted capture of known retinal dystrophy genes identified mutations in 60% of cases. This relatively high success rate may reflect enrichment for consanguineous cases in the local Yorkshire population, and the use of multiplex families. Nevertheless this is a promising high throughput approach to retinal dystrophy diagnostics.     

Frame-shifted APOBEC3A encodes two alternative proapoptotic proteins that target the mitochondrial network

The human APOBEC3A (A3A) cytidine deaminase is a powerful DNA mutator enzyme recognized as a major source of somatic mutations in tumor cell genomes. However, there is a discrepancy between APOBEC3A mRNA levels after interferon stimulation in myeloid cells and A3A detection at the protein level. To understand this difference, we investigated the expression of two novel alternative “A3Alt” proteins encoded in the +1-shifted reading frame of the APOBEC3A gene. A3Alt-L and its shorter isoform A3Alt-S appear to be transmembrane proteins targeted to the mitochondrial compartment that induce membrane depolarization and apoptosis. Thus, the APOBEC3A gene represents a new example wherein a single gene encodes two proapoptotic proteins, A3A cytidine deaminases that target the genome and A3Alt proteins that target mitochondria.     

Comparison of RNA Editing Activity of APOBEC1-A1CF and APOBEC1-RBM47 Complexes Reconstituted in HEK293T Cells

RNA editing is an important form of regulating gene expression and activity. APOBEC1 cytosine deaminase was initially characterized as pairing with a cofactor, A1CF, to form an active RNA editing complex that specifically targets APOB RNA in regulating lipid metabolism. Recent studies revealed that APOBEC1 may be involved in editing other potential RNA targets in a tissue-specific manner, and another protein, RBM47, appears to instead be the main cofactor of APOBEC1 for editing APOB RNA. In this report, by expressing APOBEC1 with either A1CF or RBM47 from human or mouse in an HEK293T cell line with no intrinsic APOBEC1/A1CF/RBM47 expression, we have compared direct RNA editing activity on several known cellular target RNAs. By using a sensitive cell-based fluorescence assay that enables comparative quantification of RNA editing through subcellular localization changes of eGFP, the two APOBEC1 cofactors, A1CF and RBM47, showed clear differences for editing activity on APOB and several other tested RNAs, and clear differences were observed when mouse versus human genes were tested. In addition, we have determined the minimal domain requirement of RBM47 needed for activity. These results provide useful functional characterization of RBM47 and direct biochemical evidence for the differential editing selectivity on a number of RNA targets.