A single transcription factor is sufficient to induce and maintain secretory cell architecture.

We hypothesized that basic helix-loop-helix (bHLH) MIST1 (BHLHA15) is a "scaling factor" that universally establishes secretory morphology in cells that perform regulated secretion. Here, we show that targeted deletion of MIST1 caused dismantling of the secretory apparatus of diverse exocrine cells. Parietal cells (PCs), whose function is to pump acid into the stomach, normally lack MIST1 and do not perform regulated secretion. Forced expression of MIST1 in PCs caused them to expand their apical cytoplasm, rearrange mitochondrial/lysosome trafficking, and generate large secretory granules. Mist1 induced a cohort of genes regulated by MIST1 in multiple organs but did not affect PC function. MIST1 bound CATATG/CAGCTG E boxes in the first intron of genes that regulate autophagosome/lysosomal degradation, mitochondrial trafficking, and amino acid metabolism. Similar alterations in cell architecture and gene expression were also caused by ectopically inducing MIST1 in vivo in hepatocytes. Thus, MIST1 is a scaling factor necessary and sufficient by itself to induce and maintain secretory cell architecture. Our results indicate that, whereas mature cell types in each organ may have unique developmental origins, cells performing similar physiological functions throughout the body share similar transcription factor-mediated architectural "blueprints."

APOBEC1 mediated C-to-U RNA editing: target sequence and trans-acting factor contribution to 177 RNA editing events in 119 murine transcripts in-vivo.

Mammalian C-to-U RNA editing was described more than 30 years ago as a single nucleotide modification in small intestinal Apob RNA, later shown to be mediated by the RNA-specific cytidine deaminase APOBEC1. Reports of other examples of C-to-U RNA editing, coupled with the advent of genome-wide transcriptome sequencing, identified an expanded range of APOBEC1 targets. Here we analyze the cis-acting regulatory components of verified murine C-to-U RNA editing targets, including nearest neighbor as well as flanking sequence requirements and folding predictions. RNA secondary structure of the editing cassette was associated with editing frequency and exhibited minimal free energy values comparable to small nuclear RNAs. We summarize findings demonstrating the relative importance of trans-acting factors (A1CF, RBM47) acting in concert with APOBEC1. Co-factor dominance was associated with editing frequency, with RNAs targeted by both RBM47 and A1CF edited at a lower frequency than RBM47 dominant targets. Using this information, we developed a multivariable linear regression model to predict APOBEC1 dependent C-to-U RNA editing efficiency, incorporating factors independently associated with editing frequencies based on 103 Sanger-confirmed editing sites, which accounted for 84% of the observed variance. This model also predicted a composite score for available human C-to-U RNA targets, which again correlated with editing frequency.

Regenerative proliferation of differentiated cells by mTORC1-dependent paligenosis.

In 1900, Adami speculated that a sequence of context-independent energetic and structural changes governed the reversion of differentiated cells to a proliferative, regenerative state. Accordingly, we show here that differentiated cells in diverse organs become proliferative via a shared program. Metaplasia-inducing injury caused both gastric chief and pancreatic acinar cells to decrease mTORC1 activity and massively upregulate lysosomes/autophagosomes; then increase damage associated metaplastic genes such as Sox9; and finally reactivate mTORC1 and re-enter the cell cycle. Blocking mTORC1 permitted autophagy and metaplastic gene induction but blocked cell cycle re-entry at S-phase. In kidney and liver regeneration and in human gastric metaplasia, mTORC1 also correlated with proliferation. In lysosome-defective Gnptab-/- mice, both metaplasia-associated gene expression changes and mTORC1-mediated proliferation were deficient in pancreas and stomach. Our findings indicate differentiated cells become proliferative using a sequential program with intervening checkpoints: (i) differentiated cell structure degradation; (ii) metaplasia- or progenitor-associated gene induction; (iii) cell cycle re-entry. We propose this program, which we term "paligenosis", is a fundamental process, like apoptosis, available to differentiated cells to fuel regeneration following injury.

Apobec1 complementation factor (A1CF) and RBM47 interact in tissue-specific regulation of C to U RNA editing in mouse intestine and liver.

Mammalian C to U RNA is mediated by APOBEC1, the catalytic deaminase, together with RNA binding cofactors (including A1CF and RBM47) whose relative physiological requirements are unresolved. Although A1CF complements APOBEC1 for in vitro RNA editing, A1cf-/- mice exhibited no change in apolipoproteinB (apoB) RNA editing, while Rbm47 mutant mice exhibited impaired intestinal RNA editing of apoB as well as other targets. Here we examined the role of A1CF and RBM47 in adult mouse liver and intestine, following deletion of either one or both gene products and also following forced (liver or intestinal) transgenic A1CF expression. There were minimal changes in hepatic and intestinal apoB RNA editing in A1cf-/- mice and no changes in either liver- or intestine-specific A1CF transgenic mice. Rbm47 liver-specific knockout (Rbm47LKO ) mice demonstrated reduced editing in a subset (11 of 20) of RNA targets, including apoB. By contrast, apoB RNA editing was virtually eliminated (<6% activity) in intestine-specific (Rbm47IKO ) mice with only five of 53 targets exhibiting C-to-U RNA editing. Double knockout of A1cf and Rbm47 in liver (ARLKO ) eliminated apoB RNA editing and reduced editing in the majority of other targets, with no changes following adenoviral APOBEC1 administration. Intestinal double knockout mice (ARIKO ) demonstrated further reduced editing (<10% activity) in four of five of the residual APOBEC1 targets identified in ARIKO mice. These data suggest that A1CF and RBM47 each function independently, yet interact in a tissue-specific manner, to regulate the activity and site selection of APOBEC1 dependent C-to-U RNA editing.

Monoacylglycerol Acyltransferase 1 Knockdown Exacerbates Hepatic Ischemia/Reperfusion Injury in Mice With Hepatic Steatosis.

Nonalcoholic fatty liver disease (NAFLD) is becoming the most common indication for liver transplantation. The growing prevalence of NAFLD not only increases the demand for liver transplantation, but it also limits the supply of available organs because steatosis predisposes grafts to ischemia/reperfusion injury (IRI) and many steatotic grafts are discarded. We have shown that monoacylglycerol acyltransferase (MGAT) 1, an enzyme that converts monoacylglycerol to diacylglycerol, is highly induced in animal models and patients with NAFLD and is an important mediator in NAFLD-related insulin resistance. Herein, we sought to determine whether Mogat1 (the gene encoding MGAT1) knockdown in mice with hepatic steatosis would reduce liver injury and improve liver regeneration following experimental IRI. Antisense oligonucleotides (ASO) were used to knockdown the expression of Mogat1 in a mouse model of NAFLD. Mice then underwent surgery to induce IRI. We found that Mogat1 knockdown reduced hepatic triacylglycerol accumulation, but it unexpectedly exacerbated liver injury and mortality following experimental ischemia/reperfusion surgery in mice on a high-fat diet. The increased liver injury was associated with robust effects on the hepatic transcriptome following IRI including enhanced expression of proinflammatory cytokines and chemokines and suppression of enzymes involved in intermediary metabolism. These transcriptional changes were accompanied by increased signs of oxidative stress and an impaired regenerative response. We have shown that Mogat1 knockdown in a mouse model of NAFLD exacerbates IRI and inflammation and prolongs injury resolution, suggesting that Mogat1 may be necessary for liver regeneration following IRI and that targeting this metabolic enzyme will not be an effective treatment to reduce steatosis-associated graft dysfunction or failure.

Parent-of-origin effects of A1CF and AGO2 on testicular germ-cell tumors, testicular abnormalities, and fertilization bias.

Testicular tumors, the most common cancer in young men, arise from abnormalities in germ cells during fetal development. Unconventional inheritance for testicular germ cell tumor (TGCT) risk both in humans and mice implicates epigenetic mechanisms. Apolipoprotein B mRNA-editing enzyme complex 1 (APOBEC1) cytidine deaminase and Deadend-1, which are involved in C-to-U RNA editing and microRNA-dependent mRNA silencing, respectively, are potent epigenetic modifiers of TGCT susceptibility in the genetically predisposed 129/Sv inbred mouse strain. Here, we show that partial loss of either APOBEC1 complementation factor (A1CF), the RNA-binding cofactor of APOBEC1 in RNA editing, or Argonaute 2 (AGO2), a key factor in the biogenesis of certain noncoding RNAs, modulates risk for TGCTs and testicular abnormalities in both parent-of-origin and conventional genetic manners. In addition, non-Mendelian inheritance was found among progeny of A1cf and Ago2 mutant intercrosses but not in backcrosses and without fetal loss. Together these findings suggest nonrandom union of gametes rather than meiotic drive or preferential lethality. Finally, this survey also suggested that A1CF contributes to long-term reproductive performance. These results directly implicate the RNA-binding proteins A1CF and AGO2 in the epigenetic control of germ-cell fate, urogenital development, and gamete functions.

A stable but reversible integrated surrogate reporter for assaying CRISPR/Cas9-stimulated homology-directed repair.

The discovery and application of CRISPR/Cas9 technology for genome editing has greatly accelerated targeted mutagenesis in a variety of organisms. CRISPR/Cas9-mediated site-specific cleavage is typically exploited for the generation of insertions or deletions (indels) after aberrant dsDNA repair via the endogenous non-homology end-joining (NHEJ) pathway or, alternatively, for enhancing homology-directed repair to facilitate the generation of a specific mutation (or "knock-in"). However, there is a need for efficient cellular assays that can measure Cas9/guide RNA activity. Reliable methods for enriching and identifying desired mutants are also lacking. Here we describe a method using the Piggybac transposon for stable genomic integration of an H2B-GFP reporter or a hygromycin resistance gene for assaying Cas9 target cleavage and homology-directed repair. The H2B-GFP fusion protein provides increased stability and an obvious pattern of nuclear localization. This method, called SRIRACCHA (i.e. a stable, but reversible, integrated reporter for assaying CRISPR/Cas-stimulated HDR activity), enables the enrichment of mutants via selection of GFP-positive or hygromycin-resistant mammalian cells (immortalized or non-immortalized) as a surrogate for the modification of the endogenous target site. Currently available hyperactive Piggybac transposase mutants allow both delivery and removal of the surrogate reporters, with minimal risk of generating undesirable mutations. This assay permits rapid screening for efficient guide RNAs and the accelerated identification of mutant clones and is applicable to many cell types. We foresee the utility of this approach in contexts in which the maintenance of genomic integrity is essential, for example, when engineering cells for therapeutic purposes.

Protocol to isolate RBP-mRNA complexes using RNA-CLIP and examine target mRNAs.

RNA-binding proteins (RBPs) regulate diverse functions by interacting with target transcripts. Here we present a protocol to isolate RBP-mRNA complexes using RNA-CLIP and examine target mRNAs in association with ribosomal populations. We describe steps to identify specific RBPs and RNA targets reflecting a variety of developmental, physiological, and pathological states. This protocol enables RNP complex isolation from tissue sources (liver and small intestine) or populations of primary cells (hepatocytes), but not at a single-cell level. For complete details on the use and execution of this protocol, please refer to Blanc et al. (2014)1 and Blanc et al. (2021).2.

APOBEC-1 deletion enhances cisplatin-induced acute kidney injury.

Cisplatin (CP) induces acute kidney injury (AKI) whereby proximal tubules undergo regulated necrosis. Repair is almost complete after a single dose. We now demonstrate a role for Apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 (Apobec-1) that is prominently expressed at the interface between acute and chronic kidney injury (CKD), in the recovery from AKI. Apobec-1 knockout (KO) mice exhibited greater mortality than in wild type (WT) and more severe AKI in both CP- and unilateral ischemia reperfusion (IR) with nephrectomy. Specifically, plasma creatinine (pCr) 2.6 ± 0.70 mg/dL for KO, n = 10 and 0.16 ± 0.02 for WT, n = 6, p < 0.0001 in CP model and 1.34 ± 0.22 mg/dL vs 0.75 ± 0.06, n = 5, p < 0.05 in IR model. The kidneys of Apobec-1 KO mice showed increased necrosis, increased expression of KIM-1, NGAL, RIPK1, ASCL4 and increased lipid accumulation compared to WT kidneys (p < 0.01). Neutrophils and activated T cells were both increased, while macrophages were reduced in kidneys of Apobec-1 KO animals. Overexpression of Apobec-1 in mouse proximal tubule cells protected against CP-induced cytotoxicity. These findings suggest that Apobec-1 mediates critical pro-survival responses to renal injury and increasing Apobec-1 expression could be an effective strategy to mitigate AKI.

RBM47 regulates intestinal injury and tumorigenesis by modifying proliferation, oxidative response, and inflammatory pathways.

RNA-binding protein 47 (RBM47) is required for embryonic endoderm development, but a role in adult intestine is unknown. We studied intestine-specific Rbm47-knockout mice (Rbm47-IKO) following intestinal injury and made crosses into ApcMin/+ mice to examine alterations in intestinal proliferation, response to injury, and tumorigenesis. We also interrogated human colorectal polyps and colon carcinoma tissue. Rbm47-IKO mice exhibited increased proliferation and abnormal villus morphology and cellularity, with corresponding changes in Rbm47-IKO organoids. Rbm47-IKO mice adapted to radiation injury and were protected against chemical-induced colitis, with Rbm47-IKO intestine showing upregulation of antioxidant and Wnt signaling pathways as well as stem cell and developmental genes. Furthermore, Rbm47-IKO mice were protected against colitis-associated cancer. By contrast, aged Rbm47-IKO mice developed spontaneous polyposis, and Rbm47-IKO ApcMin/+ mice manifested an increased intestinal polyp burden. RBM47 mRNA was decreased in human colorectal cancer versus paired normal tissue, along with alternative splicing of tight junction protein 1 mRNA. Public databases revealed stage-specific reduction in RBM47 expression in colorectal cancer associated independently with decreased overall survival. These findings implicate RBM47 as a cell-intrinsic modifier of intestinal growth, inflammatory, and tumorigenic pathways.

Intestine-specific expression of Apobec-1 rescues apolipoprotein B RNA editing and alters chylomicron production in Apobec1 -/- mice.

Intestinal apolipoprotein B (apoB) mRNA undergoes C-to-U editing, mediated by the catalytic deaminase apobec-1, which results in translation of apoB48. Apobec1(-/-) mice produce only apoB100 and secrete larger chylomicron particles than those observed in wild-type (WT) mice. Here we show that transgenic rescue of intestinal apobec-1 expression (Apobec1(Int/O)) restores C-to-U RNA editing of apoB mRNA in vivo, including the canonical site at position 6666 and also at approximately 20 other newly identified downstream sites present in WT mice. The small intestine of Apobec1(Int/O) mice produces only apoB48, and the liver produces only apoB100. Serum chylomicron particles were smaller in Apobec1(Int/O) mice compared with those from Apobec1(-/-) mice, and the predominant fraction of serum apoB48 in Apobec1(Int/O) mice migrated in lipoproteins smaller than chylomicrons, even when these mice were fed a high-fat diet. Because apoB48 arises exclusively from the intestine in Apobec1(Int/O) mice and intestinal apoB48 synthesis and secretion rates were comparable to WT mice, we were able to infer the major sites of origin of serum apoB48 in WT mice. Our findings imply that less than 25% of serum apoB48 in WT mice arises from the intestine, with the majority originating from the liver.

Apobec-1 complementation factor modulates liver regeneration by post-transcriptional regulation of interleukin-6 mRNA stability.

Apobec-1 complementation factor (ACF) is the RNA binding subunit of a core complex that mediates C to U RNA editing of apolipoprotein B (apoB) mRNA. Targeted deletion of the murine Acf gene is early embryonic lethal and Acf(-/-) blastocysts fail to implant and proliferate, suggesting that ACF plays a key role in cell growth and differentiation. Here we demonstrate that heterozygous Acf(+/-) mice exhibit decreased proliferation and impaired liver mass restitution following partial hepatectomy (PH). To pursue the mechanism of impaired liver regeneration we examined activation of interleukin-6 (IL-6) a key cytokine required for induction of hepatocyte proliferation following PH. Peak induction of hepatic IL-6 mRNA abundance post PH was attenuated >80% in heterozygous Acf(+/-) mice, along with decreased serum IL-6 levels. IL-6 secretion from isolated Kupffer cells (KC) was 2-fold greater in wild-type compared with heterozygous Acf(+/-) mice. Recombinant ACF bound an AU-rich region in the IL-6 3'-untranslated region with high affinity and IL-6 mRNA half-life was significantly shorter in KC isolated from Acf(+/-) mice compared with wild-type controls. These findings suggest that ACF regulates liver regeneration following PH at least in part by controlling the stability of IL-6 mRNA. The results further suggest a new RNA target and an unanticipated physiological function for ACF beyond apoB RNA editing.

Education by a nurse increases response of patients with chronic hepatitis C to therapy with peginterferon-α2a and ribavirin.

BACKGROUND & AIMS: Education of patients with chronic hepatitis C has been proposed to increase response to therapy with peginterferon and ribavirin. We performed a prospective study to determine the effects of systematic consultation by a nurse on patient adherence and the efficacy of therapy. METHODS: We analyzed data from 244 patients who received either systematic consultation after each medical visit from a nurse who used a standard evaluation grid and provided information about the disease and treatment (group A [GrA], n = 123) or the conventional clinical follow-up procedure (group B [GrB], n = 121). Treatment lasted 24 to 48 weeks. RESULTS: Characteristics of each group were similar at baseline, including prior treatment (42.6% in GrA and 36.0% in GrB). Overall, GrA had significantly better adherence to treatment than GrB (74.0% vs 62.8%), especially among patients who received 48 weeks of treatment (69.7% vs 53.2%; P < .03). Significantly more patients in GrA had a sustained virologic response, compared with GrB overall (38.2% vs 24.8%; P < .02), as well as treatment-naive patients (47.1% vs 30.3%; P < .05), and those with genotypes 1, 4, or 5 infections (31.6% vs 13.3%; P < .007). There were no differences between GrA and GrB in response of patients with genotypes 2 or 3 infections or advanced fibrosis. Prognostic factors for a sustained virologic response (based on bivariate and multivariate analyses) were virologic response at week 12 (odds ratio [OR], 1.9; P < .0001), genotypes 2 or 3 (OR, 2.9; P < .0001), therapeutic education (OR, 2.5; P < .02), and lack of previous treatment (OR, 2.3; P < .005). CONCLUSIONS: Therapeutic education by a specialized nurse increases the response of patients with hepatitis C to therapy, particularly in difficult-to-treat patients.

The Dance of Becoming: Pedagogy in Dance/Movement Therapy in the United States.

The purpose of this study was to begin to define pedagogical theory and practice in the field of dance/movement therapy (DMT). Fourteen DMT educators from American dance therapy association approved programs participated in the study, taking part in individual semi-structured interviews through a phenomenological lens. The participants had taught in the DMT field for at least five years and at most 44 years. Utilizing grounded theory methods, two focus groups were also conducted in which six DMT educators discussed initial qualitative themes from the individual interviews. Through an engaged process, participants were able to participate in the further defining of the study's themes. Data were analyzed using grounded theory methods of initial and focused coding. The researcher also used member checking, peer review, and a personal research journal to name her own reflexive position within the emerging data. The researcher's findings centered around six qualitative themes. These themes named the importance of the DMT student's development of self-awareness including body identity, cultural identity, and professional identity all housed within the experience of embodied learning. Findings also named the importance of educator transparency and modeling in the classroom to create space for student exploration. Recommendations from the study aimed towards creating more opportunities for educators to collaborate and communicate across the field with the goal of creating best practices for DMT education. Also recommendation for DMT educators centered around clarity of expectations in the embodied self-reflective learning process.

Apobec1 complementation factor overexpression promotes hepatic steatosis, fibrosis, and hepatocellular cancer.

The RNA-binding protein Apobec1 complementation factor (A1CF) regulates posttranscriptional ApoB mRNA editing, but the range of RNA targets and the long-term effect of altered A1CF expression on liver function are unknown. Here we studied hepatocyte-specific A1cf-transgenic (A1cf+/Tg), A1cf+/Tg Apobec1-/-, and A1cf-/- mice fed chow or high-fat/high-fructose diets using RNA-Seq, RNA CLIP-Seq, and tissue microarrays from human hepatocellular cancer (HCC). A1cf+/Tg mice exhibited increased hepatic proliferation and steatosis, with increased lipogenic gene expression (Mogat1, Mogat2, Cidea, Cd36) associated with shifts in polysomal RNA distribution. Aged A1cf+/Tg mice developed spontaneous fibrosis, dysplasia, and HCC, and this development was accelerated on a high-fat/high-fructose diet and was independent of Apobec1. RNA-Seq revealed increased expression of mRNAs involved in oxidative stress (Gstm3, Gpx3, Cbr3), inflammatory response (Il19, Cxcl14, Tnfα, Ly6c), extracellular matrix organization (Mmp2, Col1a1, Col4a1), and proliferation (Kif20a, Mcm2, Mcm4, Mcm6), and a subset of mRNAs (including Sox4, Sox9, Cdh1) were identified in RNA CLIP-Seq. Increased A1CF expression in human HCC correlated with advanced fibrosis and with reduced survival in a subset with nonalcoholic fatty liver disease. In conclusion, we show that hepatic A1CF overexpression selectively alters polysomal distribution and mRNA expression, promoting lipogenic, proliferative, and inflammatory pathways leading to HCC.

Decreased expression of cholesterol 7alpha-hydroxylase and altered bile acid metabolism in Apobec-1-/- mice lead to increased gallstone susceptibility.

Quantitative trait mapping in mice identified a susceptibility locus for gallstones (Lith6) spanning the Apobec-1 locus, the structural gene encoding the RNA-specific cytidine deaminase responsible for production of apolipoprotein B48 in mammalian small intestine and rodent liver. This observation prompted us to compare dietary gallstone susceptibility in Apobec-1(-/-) mice and congenic C57BL/6 wild type controls. When fed a lithogenic diet (LD) for 2 weeks, 90% Apobec-1(-/-) mice developed solid gallstones in comparison with 16% wild type controls. LD-fed Apobec-1(-/-) mice demonstrated increased biliary cholesterol secretion as well as increased cholesterol saturation and bile acid hydrophobicity indices. These changes occurred despite a relative decrease in cholesterol absorption in LD-fed Apobec-1(-/-) mice. Among the possible mechanisms to account for this phenotype, expression of Cyp7a1 mRNA and protein were significantly decreased in chow-fed Apobec-1(-/-) mice, decreasing further in LD-fed animals. Cyp7a1 transcription in hepatocyte nuclei, however, was unchanged in Apobec-1(-/-) mice, excluding transcriptional repression as a potential mechanism for decreased Cyp7a1 expression. We demonstrated that APOBEC-1 binds to AU-rich regions of the 3'-untranslated region of the Cyp7a1 transcript, containing the UUUN(A/U)U consensus motif, using both UV cross-linking to recombinant APOBEC-1 and in vivo RNA co-immunoprecipitation. In vivo Apobec-1-dependent modulation of Cyp7a1 expression was further confirmed following adenovirus-Apobec-1 administration to chow-fed Apobec-1(-/-) mice, which rescued Cyp7a1 gene expression. Taken together, the findings suggest that the AU-rich RNA binding-protein Apobec-1 mediates post-transcriptional regulation of murine Cyp7a1 expression and influences susceptibility to diet-induced gallstone formation.

Deletion of the AU-rich RNA binding protein Apobec-1 reduces intestinal tumor burden in Apc(min) mice.

The RNA-specific cytidine deaminase apobec-1 is an AU-rich RNA binding protein that binds the 3' untranslated region (UTR) of cyclooxygenase-2 (Cox-2) mRNA and stabilizes its turnover in vitro. Cox-2 overexpression accompanies intestinal adenoma formation in both humans and mice. Evidence from both genetic deletion studies as well as from pharmacologic inhibition has implicated Cox-2 in the development of intestinal adenomas in experimental animals and in adenomas and colorectal cancer in humans. Here, we show that small intestinal adenoma formation is dramatically reduced in compound Apc(min/+) apobec-1(-/-) mice when compared with the parental Apc(min/+) strain. This reduced tumor burden was found in association with increased small intestinal apoptosis and reduced proliferation in small intestinal crypt-villus units from compound Apc(min/+) apobec-1(-/-) mice. Intestinal adenomas from compound Apc(min/+) apobec-1(-/-) mice showed a <2-fold increase in Cox-2 mRNA abundance and reduced prostaglandin E(2) content compared with adenomas from the parental Apc(min/+) strain. In addition, there was reduced expression in adenomas from compound Apc(min/+) apobec-1(-/-) mice of other mRNAs (including epidermal growth factor receptor, peroxisome proliferator-activated receptor delta, prostaglandin receptor EP4, and c-myc), each containing the apobec-1 consensus binding site within their 3'-UTR. Adenovirus-mediated apobec-1 introduction into HCA-7 (colorectal cancer) cells showed a dose-dependent increase in Cox-2 protein and stabilization of endogenous Cox-2 mRNA. These findings suggest that deletion of apobec-1, by modulating expression of AU-rich RNA targets, provides an important mechanism for attenuating a dominant genetic restriction point in intestinal adenoma formation.

Targeted deletion of the murine apobec-1 complementation factor (acf) gene results in embryonic lethality.

apobec-1 complementation factor (ACF) is an hnRNP family member which functions as the obligate RNA binding subunit of the core enzyme mediating C-to-U editing of the nuclear apolipoprotein B (apoB) transcript. ACF binds to both apoB RNA and apobec-1, the catalytic cytidine deaminase, which then results in site-specific posttranscriptional editing of apoB mRNA. Targeted deletion of apobec1 eliminates C-to-U editing of apoB mRNA but is otherwise well tolerated. However, the functions and potential targets of ACF beyond apoB mRNA editing are unknown. Here we report the results of generating acf knockout mice using homologous recombination. While heterozygous acf(+/)(-) mice were apparently healthy and fertile, no viable acf(-)(/)(-) mice were identified. Mutant acf(-)(/)(-) embryos were detectable only until the blastocyst (embryonic day 3.5 [E3.5]) stage. No acf(-)(/)(-) blastocysts were detectable following implantation at E4.5, and isolated acf(-)(/)(-) blastocysts failed to proliferate in vitro. Small interfering RNA knockdown of ACF in either rat (apobec-1-expressing) or human (apobec-1-deficient) hepatoma cells decreased ACF protein expression and induced a commensurate increase in apoptosis. Taken together, these data suggest that ACF plays a crucial role, which is independent of apobec-1 expression, in cell survival, particularly during early embryonic development.

Mouse models as tools to explore cytidine-to-uridine RNA editing.

RNA editing is a process through which the nucleotide sequence specified in the genomic template is modified to produce a different nucleotide sequence in the transcript. RNA editing is an important mechanism of genetic regulation that amplifies genetic plasticity by allowing the production of alternative protein products from a single gene. There are two generic classes of RNA editing in nuclei, involving enzymatic deamination of either C-to-U or A-to-I nucleotides. The best characterized example of C-to-U RNA editing is that of apolipoprotein B (apoB), which is mediated by a holoenzyme that contains a minimal core composed of an RNA-specific cytidine deaminase apobec-1, and its cofactor apobec-1 complementation factor (ACF). C-to-U editing of apoB RNA generates two different isoforms--apoB100 and apoB48--from a single transcript. Both are important regulators of lipid transport and metabolism, and are functionally distinct. C-to-U apoB RNA editing is regulated by a range of factors including developmental, nutritional, environmental, and metabolic stimuli. Rodent models have provided a tractable system in which to study the effects of such stimuli on lipid metabolism. In addition, both transgenic and gene knockout experiments have provided important insights into gain and loss of function approaches for studying C-to-U RNA editing in a murine background.