Molecular cloning of a human small intestinal apolipoprotein B mRNA editing protein

Abstract

Mammalian small intestinal apolipoprotein B (apo B) mRNA undergoes posttranscriptional cytidine deamination with the production of an In frame stop codon and the translation of apo B48. We have isolated a cDNA from human jejunum which mediates In vitro editing of a synthetic apo B RNA template upon compiementation with chicken Intestinal S100 extracts.

Identification of domains in apobec-1 complementation factor required for RNA binding and apolipoprotein-B mRNA editing

“…Summary of the data from N-and C-terminal deletion mutants of ACF+ The structure of wild-type ACF is shown at the top+ The three RRM domains are indicated as RRM1, RRM2, and RRM3+ The hatched and stippled bars represent the RG-rich region and the putative double-stranded RNA-binding domain, respectively+ A diagram of key N-and C-terminal deletion mutants is shown on the left+ All mutants were tested in at least three independent experiments, and the results were quantified by PhosphorImager analysis+ The results for complementing activity are expressed relative to the activity of the full-length ACF (average % 6 SD)+ The K d s for apo-B mRNA were obtained from filter-binding experiments and were calculated as the protein concentration at which 50% of the RNA was bound (average nM 6 SD)+ The results from the EMSA assays and apobec-1 binding assays are expressed qualitatively (ϩ or Ϫ)+ ND: not determined+ 76 A. Mehta and D.M. Driscoll text of the holoenzyme+ In contrast to ACF, apobec-1 has a weak nonspecific RNA-binding activity (Anant et al+, 1995;Navaratnam et al+, 1995) with a K d for apo-B mRNA of ;450 nM (Anant & Davidson, 2000)+ We found that the addition of recombinant apobec-1 did not change the affinity of ACF for apo-B mRNA (S+ Murata & D+M+ Driscoll, unpubl+ observations), which suggests that the recognition of apo-B mRNA by the holoenzyme is mediated solely by ACF+ This result should be interpreted cautiously, because all of the recombinant proteins may not assemble into an active enzyme complex+ In addition to our in vitro studies, we present evidence that suggests that ACF is associated with apo-B mRNA in vivo in HepG2 cells, a human hepatoma cell line that does not express apobec-1+ In terms of physiological relevance, apo-B mRNA and ACF are both expressed in the livers of primates and rabbits, even though these tissues lack apobec-1 and are not competent to edit (Greeve et al+, 1993;Giannoni et al+, 1994;Hadjiagapiou et al+, 1994)+ Our results raise the possibility that ACF has an additional function in the processing of apo-B mRNA that is independent of editing+ In fact, ACF may play a more general role in mRNA metabolism, as ACF is widely expressed in other tissues that do not express apo-B mRNA (Mehta et al+, 2000)+ This question is of particular interest because it was recently suggested that apobec-1 is involved in FIGURE 7. Mutagenesis of the RRM domains in ACF+ A: Point mutations in the conserved RNP2 and RNP1 motifs were made in the three RRMs of human ACF as indicated+ The mutants were expressed in bacteria as Strep-tagged proteins and analyzed for their ability to bind to 32 P-labeled apo-B RNA in a filter-binding assay as described in Figure 2A+ The calculated K d values are discussed in the text+ B: RRM deletion mutants were analyzed in a filter-binding assay and the K d s for apo-B mRNA were calculated as described in the legend to Figure 6+ the stabilization of mRNAs that undergo rapid degradation, including c-myc mRNA (Anant & Davidson, 2000)+ Experiments are currently in progress to identify other mRNA targets that are bound to ACF in vivo+ In many multi-RRM proteins, the RRM domains mediate RNA recognition whereas the auxiliary domain ...…”

APOBEC1-Mediated Editing and Attenuation of Herpes Simplex Virus 1 DNA Indicate That Neurons Have an Antiviral Role during Herpes Simplex Encephalitis

“…However, we believe that this is unlikely, since the expression profile of human A1 is quite distinct from that of rodent A1. Rat A1 has at least three clusters of different transcriptional promoter sites within the gene giving rise to its diverse tissue expression, whereas human A1 appears to be confined to the small intestine, with no evidence of its expression in other tissues (20,26). Nonetheless, it will be interesting to explore whether human A1 is expressed in neurons during HSV-1 infection.…”

Biochemical and molecular characterization of two cytidine deaminases in the nematode Caenorhabditis elegans

“…The localization of the CDDs to the intestinal cells of C. elegans is an interesting observation, particularly since apobec-1 is restricted to epithelial cells lining the gastrointestinal tract in humans [38]. The gut in C. elegans is an extremely active tissue, and the presence of CDDs in the cells of the intestine might reflect the requirement for nucleic acid synthesis in this compartment, and a role for CDD in the salvage pathway.…”