Naveenan Navaratnam

The apolipoprotein B mRNA editing complex performs a multifunctional cycle and suppresses nonsense-mediated decay

The C to U editing of apolipoprotein B (apoB) mRNA is mediated by a minimal complex composed of an RNA‐binding cytidine deaminase (APOBEC1) and a complementing specificity factor (ACF). This editing generates a premature termination codon and a truncated open reading frame. We demonstrate that the APOBEC1—ACF holoenzyme mediates a multifunctional cycle. The atypical APOBEC1 nuclear localization signal is involved in RNA binding and is used to import ACF into the nucleus as cargo. APOBEC1 alone induces nonsense‐mediated decay (NMD). The APOBEC1—ACF complex edits and remains associated with the edited RNA to protect it from NMD. The APOBEC1 nuclear export signal is involved in the export of ACF and the edited apoB mRNA together, to the site of translation.

RNA editing: cytidine to uridine conversion in apolipoprotein B mRNA.

RNA editing is a post-transcriptional process that changes the informational capacity within the RNA. These processes include alterations made by nucleotide deletion, insertion and base conversion. A to I and C to U conversion occurs in mammals and these editing events are catalysed by RNA binding deaminases. C to U editing of apoB mRNA was the first mammalian editing event to be identified. The minimal protein complex necessary for apoB mRNA editing has been determined and consists of APOBEC-1 and ACF. Overexpression of APOBEC-1 in transgenic animals caused liver dysplasia and APOBEC-1 has been identified in neurofibromatosis type 1 tumours, suggesting that RNA editing may be another mechanism for tumourigenesis. Several APOBEC-1-like proteins have been identified, including a family of APOBEC-1-related proteins with unknown function on chromosome 22. This review summarises the different types of RNA editing and discusses the current status of C to U apoB mRNA editing. This knowledge is very important in understanding the structure and function of these related proteins and their role in biology.

An Overview of Cytidine Deaminases

Enzymes that deaminate cytidine to uridine play an important role in a variety of pathways from bacteria to man.Ancestral members of this family were able to deaminate cytidine only in a mononucleotide or nucleoside context. Recently, a family of enzymes has been discovered with the ability to deaminate cytidines on RNA or DNA. The first member of this new family is APOBEC1, which deaminates apolipoprotein B messenger RNA to generate a premature stop codon. APOBEC1 has the conserved active site motif found in Escherichia coli cytidine deaminase. In addition, APOBEC1 has a unique motif containing 2 phenylalanine residues and an insert of 4 amino acid residues across the active site motif. This motif is present in APOBEC family members including activation-induced cytidine deaminase (AID), APOBEC2, and APOBEC3A through APOBEC3G. AID is essential for initiating class-switch recombination, somatic hypermutation, and gene conversion. The APOBEC3 family is unique to primates. APOBEC3G is able to protect cells from human immunodeficiency virus and other viral infections.This function is not unique to APOBEC3G; other APOBEC3 family members also have this ability. Overexpression of enzymes in this family can cause cancer, suggesting that the genes for the APOBEC family of proteins are proto-oncogenes. Recent advances in the understanding of the mechanism of action of this family are summarized in this review.

Arkadia Enhances Nodal/TGF-β Signaling by Coupling Phospho-Smad2/3 Activity and Turnover

Regulation of transforming growth factor-β (TGF-β) signaling is critical in vertebrate development, as several members of the TGF-β family have been shown to act as morphogens, controlling a variety of cell fate decisions depending on concentration. Little is known about the role of intracellular regulation of the TGF-β pathway in development. E3 ubiquitin ligases target specific protein substrates for proteasome-mediated degradation, and several are implicated in signaling. We have shown that Arkadia, a nuclear RING-domain E3 ubiquitin ligase, is essential for a subset of Nodal functions in the embryo, but the molecular mechanism of its action in embryonic cells had not been addressed. Here, we find that Arkadia facilitates Nodal signaling broadly in the embryo, and that it is indispensable for cell fates that depend on maximum signaling. Loss of Arkadia in embryonic cells causes nuclear accumulation of phospho-Smad2/3 (P-Smad2/3), the effectors of Nodal signaling; however, these must be repressed or hypoactive as the expression of their direct target genes is reduced or lost. Molecular and functional analysis shows that Arkadia interacts with and ubiquitinates P-Smad2/3 causing their degradation, and that this is via the same domains required for enhancing their activity. Consistent with this dual function, introduction of Arkadia in homozygous null (−/−) embryonic stem cells activates the accumulated and hypoactive P-Smad2/3 at the expense of their abundance. Arkadia−/− cells, like Smad2−/− cells, cannot form foregut and prechordal plate in chimeras, confirming this functional interaction in vivo. As Arkadia overexpression never represses, and in some cells enhances signaling, the degradation of P-Smad2/3 by Arkadia cannot occur prior to their activation in the nucleus. Therefore, Arkadia provides a mechanism for signaling termination at the end of the cascade by coupling degradation of P-Smad2/3 with the activation of target gene transcription. This mechanism can account for achieving efficient and maximum Nodal signaling during embryogenesis and for rapid resetting of target gene promoters allowing cells to respond to dynamic changes in extracellular signals.

Regulation of ploidy and senescence by the AMPK‐related kinase NUAK1

Senescence is an irreversible cell‐cycle arrest that is elicited by a wide range of factors, including replicative exhaustion. Emerging evidences suggest that cellular senescence contributes to ageing and acts as a tumour suppressor mechanism. To identify novel genes regulating senescence, we performed a loss‐of‐function screen on normal human diploid fibroblasts. We show that downregulation of the AMPK‐related protein kinase 5 (ARK5 or NUAK1) results in extension of the cellular replicative lifespan. Interestingly, the levels of NUAK1 are upregulated during senescence whereas its ectopic expression triggers a premature senescence. Cells that constitutively express NUAK1 suffer gross aneuploidies and show diminished expression of the genomic stability regulator LATS1, whereas depletion of NUAK1 with shRNA exerts opposite effects. Interestingly, a dominant‐negative form of LATS1 phenocopies NUAK1 effects. Moreover, we show that NUAK1 phosphorylates LATS1 at S464 and this has a role in controlling its stability. In summary, our work highlights a novel role for NUAK1 in the control of cellular senescence and cellular ploidy.

Secondary Structure for the Apolipoprotein B mRNA Editing Site AU-BINDING PROTEINS INTERACT WITH A STEM LOOP

The C to U editing of apolipoprotein B (apoB) mRNA converts a glutamine codon in apoB100 mRNA into a stop translation codon thereby generating apoB48. The catalytic subunit of the editing enzyme, APOBEC-1, is an RNA-binding cytidine deaminase that requires auxiliary factors for the editing of apoB mRNA. Computer modeling and ribonuclease probing of the wild-type and mutant apoB RNA substrates reveal a stem loop at the editing site. This structure incorporates the essential sequence motifs required for editing. The localization of the edited cytidine within the loop suggests how it could be presented to the active site of APOBEC-1 for deamination. We have identified 43/45 kDa proteins from chick enterocytes and show evidence for their involvement in auxiliary editing activity. p43/45 demonstrates preferential binding to AU-rich RNA and to the Caauuug motif that forms the loop and proximal stem of the apoB mRNA.

Characterization of the apolipoprotein B mRNA editing enzyme: no similarity to the proposed mechanism of RNA editing in kinetoplastid protozoa.

Intestinal apolipoprotein B mRNA is edited at nucleotide 6666 by a C to U transition resulting in a translational stop codon. The enzymatic properties of the editing activity were characterised in vitro using rat enterocyte cytosolic extract. The editing activity has no nucleotide or ion cofactor requirement. It shows substrate saturation with an apparent Km for the RNA substrate of 2.2 nM. The editing enzyme requires no lag period prior to catalysis, and does not assemble into a higher order complex on the RNA substrate. In crude cytosolic extract editing activity is completely abolished by treatment with micrococcal nuclease or RNAse A. Partially purified editing enzyme is no longer sensitive to nucleases, but is inhibited in a dose dependent manner by nuclease inactivated crude extract. The buoyant density of partially purified editing enzyme is 1.3 g/ml, that of pure protein. Therefore, the apolipoprotein B mRNA editing activity consists of a well defined enzyme with no RNA component. The nuclease sensitivity in crude cytosolic extract is explained by the generation of inhibitors for the editing enzyme. The editing of apo B mRNA has little similarity to complex mRNA processing events such as splicing and unlike editing in kinetoplastid protozoa does not utilise guide RNAs.

APOBEC3B-Mediated Cytidine Deamination Is Required for Estrogen Receptor Action in Breast Cancer

Summary Estrogen receptor α (ERα) is the key transcriptional driver in a large proportion of breast cancers. We report that APOBEC3B (A3B) is required for regulation of gene expression by ER and acts by causing C-to-U deamination at ER binding regions. We show that these C-to-U changes lead to the generation of DNA strand breaks through activation of base excision repair (BER) and to repair by non-homologous end-joining (NHEJ) pathways. We provide evidence that transient cytidine deamination by A3B aids chromatin modification and remodelling at the regulatory regions of ER target genes that promotes their expression. A3B expression is associated with poor patient survival in ER+ breast cancer, reinforcing the physiological significance of A3B for ER action.

Intracellular Localization of Human Cytidine Deaminase IDENTIFICATION OF A FUNCTIONAL NUCLEAR LOCALIZATION SIGNAL

The cytidine deaminases belong to the family of multisubunit enzymes that catalyze the hydrolytic deamination of their substrate to a corresponding uracil product. They play a major role in pyrimidine nucleoside and nucleotide salvage. The intracellular distribution of cytidine deaminase and related enzymes has previously been considered to be cytosolic. Here we show that human cytidine deaminase (HCDA) is present in the nucleus. A highly specific, affinity purified polyclonal antibody against HCDA was used to analyze the intracellular localization of native HCDA in a variety of mammalian cells by in situ immunochemistry. Native HCDA was found to be present in the nucleus as well as the cytoplasm in several cell types. Indirect immunofluorescence microscopy indicated a predominantly nuclear localization of FLAG-tagged HCDA overexpressed in these cells. We have identified an amino-terminal bipartite nuclear localization signal that is both necessary and sufficient to direct HCDA and a non-nuclear reporter protein to the nucleus. We also show HCDA binding to the nuclear import receptor, importin α. Similar putative bipartite nuclear localization sequences are found in other cytidine/deoxycytidylate deaminases. The results presented here suggest that the pyrimidine nucleotide salvage pathway may operate in the nucleus. This localization may have implications in the regulation of nucleoside and nucleotide metabolism and nucleic acid biosynthesis.

The apolipoprotein B messenger RNA editing enzyme.

The editing of apolipoprotein (apo)B messenger RNA (mRNA) involves a novel C to U modification, which creates an in-frame stop-translation codon, thereby generating the carboxyl-terminal of apoB48. The 27 kDa catalytic subunit of the editing enzyme has been cloned and established to be a zinc-containing cytidine deaminase. The catalytic subunit is guided to the editing site by a second targeting subunit or subunits. A candidate for the targeting subunit is a 60 kDa protein that can be UV crosslinked to the sequence UGAU, which is part of a motif downstream of the editing site that is essential for editing.