An in vitro system for the editing of apolipoprotein B mRNA

A novel form of RNA editing generates two forms of apolipoprotein B (apo-B) mRNA by converting C at nucleotide 6666 to U or a U-like base. We have established an in vitro system for the editing of apo-B mRNA using synthetic RNAs and S100 extracts from rat hepatoma cells. Editing was detected by a sensitive primer extension assay and confirmed by DNA sequencing. The in vitro editing activity is specific and sensitive to proteinase K. Apo-B100 RNAs were synthesized in vitro from deletion mutants spanning nucleotide 6666. Synthetic RNAs containing 2383, 483, and 55 nucleotides of apo-B mRNA sequence were edited in vitro with similar efficiency, but an RNA containing 26 nucleotides was not edited.     

Quantitation of endogenous liver apolipoprotein B mRNA editing

The mRNA for apolipoprotein B is translated into either a high molecular weight (apo BH) or low molecular weight (apo BL) form of the protein depending on a novel form of RNA processing known as RNA editing. Apo BH mRNA editing is both tissue-specific and hormonally regulated and involves transition of cytidine to uridine at codon 2153 thereby converting a glutamine codon (CAA) to a translational stop codon (UAA). Three methods for quantitating the endogenous levels of liver apo B mRNA editing were compared: (1) Southern blot hybridization with discriminative thermal washes, (2) competimer-hybridization with discriminative thermal washes and (3) competimer-polymerase chain reaction (competimer-PCR). The data suggest that hybridization and PCR can yield similar quantitation when competing oligonucleotides are used. Based on competimer-PCR it is proposed that 40% and 85% of normal rat liver and small intestine apo B mRNA (respectively) are edited.     

Site-specific creation of uridine from cytidine in apolipoprotein B mRNA editing.

Human apolipoprotein (apo) B mRNA is edited in a tissue specific reaction, to convert glutamine codon 2153 (CAA) to a stop translation codon. The RNA editing product templates and hybridises as uridine, but the chemical nature of this reaction and the physical identity of the product are unknown. After editing in vitro of [32P] labelled RNA, we are able to demonstrate the production of uridine from cytidine; [alpha 32P] cytidine triphosphate incorporated into RNA gave rise to [32P] uridine monophosphate after editing in vitro, hydrolysis with nuclease P1 and thin layer chromatography using two separation systems. By cleaving the RNA into ribonuclease T1 fragments, we show that uridine is produced only at the authentic editing site and is produced in quantities that parallel an independent primer extension assay for editing. We conclude that apo B mRNA editing specifically creates a uridine from a cytidine. These observations are inconsistent with the incorporation of a uridine nucleotide by any polymerase, which would replace the alpha-phosphate and so rule out a model of endonucleolytic excision and repair as the mechanism for the production of uridine. Although transamination and transglycosylation remain to be formally excluded as reaction mechanisms our results argue strongly in favour of the apo B mRNA editing enzyme as a site-specific cytidine deaminase.     

A 40 kilodalton rat liver nuclear protein binds specifically to apolipoprotein B mRNA around the RNA editing site.

Apolipoprotein (apo) B-48 mRNA is the product of RNA editing which consists of a C----U conversion changing a CAA codon encoding Gln-2153 in apoB-100 mRNA to a UAA stop codon in apoB-48 mRNA. In the adult rat, RNA editing occurs both in the small intestine and the liver. We have studied the ability of rat liver nuclear extracts to bind to synthetic apoB mRNA segments spanning the editing site. Using an RNA gel mobility shift assay, we found the sequence-specific binding of a protein(s) to a 65-nucleotide apoB-100 mRNA. UV crosslinking followed by T1 ribonuclease digestion and SDS-polyacrylamide gel electrophoresis demonstrated the formation of a 40 kDa protein-RNA complex when 32P-labeled apoB-100 mRNA was incubated with a rat liver nuclear extract but not with HeLa nuclear extract. Binding was specific for the sense strand of apoB mRNA, and was not demonstrated with single-stranded apoB DNA, or antisense apoB RNA. The complex also failed to form if SDS was present during the UV light exposure. Binding experiments using synthetic apoB mRNAs indicate that the 40 kDa protein would also bind to apoB-48 mRNA but not apoA-I, apoA-IV, apoC-II or apoE mRNA. Experiments using deletion mutants of apoB-100 mRNA indicate efficient binding of wildtype 65-nucleotide (W65), 40-nucleotide (W40) and 26-nucleotide (W26) apoB-100 mRNA segments, but not 10-nucleotide (or smaller) segments of apoB-100 mRNA to the 40 kDa protein. In contrast, two other regions of apoB-100 mRNA, B-5' (bases 1128-3003) and B-3' (bases 11310-11390), failed to bind to the protein. The 40 kDa sequence-specific binding protein in rat liver nuclear extract may play a role in apoB-100 mRNA editing.     

Disproportionate relationship between APOBEC-1 expression and apolipoprotein B mRNA editing activity.

Apolipoprotein B (apoB) mRNA editing is a site-specific (nucleotide 6666) cytidine to uridine transition catalyzed by a cytidine deaminase, APOBEC-1, in the context of a multiprotein complex referred to as the C/U editosome. This report quantifies for the first time the effect of altering APOBEC-1 protein abundance on the proportion of edited apoB mRNAs using transfected McArdle rat hepatoma cells which had been sorted by flow cytometry into populations expressing different levels of green fluorescent protein-APOBEC-1 chimera, GFP-APOBEC. A correlation was observed in which increased expression of GFP-APOBEC protein resulted in a higher proportion of edited apoB mRNA. The number of enzyme molecules required to increase the proportion of edited apoB RNAs was disproportionately high relative to that which might have been predicted from a typical catalytic relationship. Moreover, editing of apoB mRNA at inappropriate sites (promiscuous editing) occurred in response to overexpressing GFP-APOBEC. The data suggest that experimental manipulation of APOBEC-1 abundance in the absence of other regulatory considerations will always result in some level of promiscuous editing. Coordinate expression of APOBEC-1 and the auxiliary proteins and/or regulation of their interactions may be required to increase editing activity without losing editing-site fidelity.     

Induction of RNA editing at heterologous sites by sequences in apolipoprotein B mRNA.

An RNA editing mechanism modifies apolipoprotein B (apo-B) mRNA in the intestine by converting cytosine at nucleotide (nt) 6666 to uracil. To define the sequence requirements for editing, mutant apo-B RNAs were analyzed for the ability to be edited in vitro by enterocyte extracts. Editing was detected by a sensitive and linear primer extension assay. An upstream region (nt 6648 to 6661) which affected the efficiency of editing was identified. RNAs with mutations in this efficiency sequence were edited at 22 to 160% of wild-type levels. Point mutations in a downstream 11-nt mooring sequence (nt 6671 to 6681) abolished editing, confirming previous studies (R. R. Shah, T. J. Knott, J. E. Legros, N. Navaratnam, J. C. Greeve, and J. Scott, J. Biol. Chem. 266:16301-16304, 1991). The optimal distance between the editing site and the mooring sequence is 5 nt, but a C positioned 8 nt upstream is edited even when nt 6666 contains U. The efficiency and mooring sequences were inserted individually and together adjacent to a heterologous C in apo-B mRNA. The mooring sequence alone induced editing of the C at nt 6597 both in vitro and in transfected rat hepatoma cells. Editing at nt 6597 was specific, was independent of editing at nt 6666, and was stimulated to wild-type levels when the efficiency sequence was also inserted. Introduction of the mooring sequence into a heterologous mRNA, luciferase mRNA, induced editing of an upstream cytidine. Although UV cross-linking studies have previously shown that proteins of 60 to 66 kDa cross-link to apo-B mRNA, these proteins did not cross-link to the luciferase translocation mutants.     

Detection of apolipoprotein B mRNA editing by peptide nucleic acid mediated PCR clamping.

Apolipoprotein B (apoB) mRNA editing leads to a single base change in its mRNA and the production of apoB-48. Currently, the degree of apoB mRNA editing is analyzed by the RT-PCR primer extension method. While this method is quantitative, it is labor intensive, utilizes radioactivity for labeling and may not be sensitive enough to discriminate between low levels of editing and inherent assay background levels. Peptide nucleic acid (PNA) oligonucletides have been used in single point mutation detection through PCR clamping. In the present work, we developed a PCR based assay which can detect the single base change responsible for the apoB-48 production. We found that as low as 0.5% of the edited form can be clearly detected by PNA mediated PCR clamping. When combined with the primer extension assay, an approximately 180-fold enrichment of the edited percentage is observed, reflecting selected PCR amplification of templates containing the edited base.     

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.