apoB RNA Research Articles

In vitro apolipoprotein B mRNA editing: identification of a 27S editing complex.

Specific apolipoprotein B (apoB) mRNA editing can be performed in vitro on apoB RNA substrates. Native gels and glycerol gradient sedimentation have been used to determine the physical properties of the in vitro editing activity in rat liver cytosolic S100 extracts. ApoB RNA substrates were progressively assembled as 27S complexes for 3 hr with similar kinetics as seen for the accumulation of edited RNA. Assembly was not observed on RNAs from apoB deletion constructs that did not support editing. The 27S complex contained both edited and unedited RNA sequences. Inhibition of 27S complex assembly by vanadyl-ribonucleoside complexes was accompanied by inhibition of editing. Based on these data, we propose that the 27S complex is the in vitro "editosome," A "mooring sequence" model for RNA recognition and editosome assembly has been proposed involving RNA sequences flanking the edited nucleotide.

Characterization of the apolipoprotein B mRNA editing activity in enterocyte extracts.

Apolipoprotein B (apoB) circulates in human plasma as two isoforms, apoB-100 (512 kDa) and apoB-48 (242 kDa). ApoB-48 is generated by a novel RNA editing mechanism which post-transcriptionally modifies apoB mRNA in the intestine by converting cytidine at nucleotide 6666 to uridine. This converts codon 2153 from glutamine (CAA) to a premature stop codon (UAA). To characterize the activity which edits apoB mRNA, extracts were prepared from enterocytes isolated from baboon small intestine. These extracts efficiently edit synthetic apoB RNA in vitro. Editing was detected by primer extension, and the specificity of the reaction was confirmed by DNA sequencing. Extracts prepared from other baboon tissues did not edit apoB RNA in vitro. The editing activity was partially purified by chromatography of the enterocyte extracts on DEAE-cellulose. The activity is sensitive to proteinase K but resistant to micrococcal nuclease and has an average molecular mass of 125 kDa when analyzed by gel filtration chromatography.

Apolipoprotein B mRNA editing. Direct determination of the edited base and occurrence in non-apolipoprotein B-producing cell lines.

In humans, apolipoprotein (apo) B48 is synthesized in the intestine as an obligatory constituent of chylomicrons. Apolipoprotein B48 is identical to the amino-terminal 2152 amino acids (240 kDa) of apoB100 and is translated from an edited apoB mRNA in which codon 2153 has been converted from glutamine (CAA) to what is recognized as a premature stop codon (UAA). To determine whether the apoB mRNA editing in fact converts cytosine 6666 in codon 2153 to uracil, we incubated a synthetic apoB RNA containing 32P-labeled cytosines in an in vitro editing system prepared from rabbit enterocytes. The in vitro edited RNA was purified and digested to nucleoside 5'-monophosphates, which were analyzed on two-dimensional thin-layer chromatography. We found that the edited base co-migrated with authentic uridine 5'-monophosphate. Thus, cytosine 6666 is converted to uracil, most likely by a nucleotide-specific cytosine deaminase. To determine whether apoB mRNA editing occurs in cell lines that do not synthesize apoB, we stably transfected a high expression vector containing 354 base pairs of apoB sequence into 18 different cell lines. We found apoB mRNA editing activity in five osteosarcoma cell lines and one epidermoid cell line, none of which synthesizes any detectable apoB. Thus, apoB mRNA editing occurs in cell lines that do not synthesize apoB, which suggests that mRNA editing may be a common biological phenomenon in eukaryotic cells.

Fasting decreases apolipoprotein B mRNA editing and the secretion of small molecular weight apoB by rat hepatocytes: evidence that the total amount of apoB secreted is regulated post-transcriptionally

Two different molecular weight forms of apoB are produced from a common initial transcript via editing of a Gln codon (CAA) to a stop codon (UAA), leading to a truncated translation product (apo BS) that consists of the amino terminal half of the larger form (apoBL). Previous studies have shown that fasting coordinately decreases lipogenesis and the secretion of very low density lipoprotein (VLDL) lipids and apoBS. Secretion of the apoBL is unaffected by fasting. We studied whether editing of apoB RNA is repressed by fasting, thus accounting for the selective decreased secretion of apoBS. Column chromatography of [35S]methionine-labeled lipoproteins secreted by hepatocytes from fed rats showed that essentially all of apoBL is secreted in the VLDL fraction, whereas a significant amount (15%) of apoBS is secreted associated as lipoproteins eluting in the HDL fractions. Fasting decreased the relative amount of apoBS that eluted in the VLDL fractions and increased the amount secreted in the HDL fractions. Consistent with previous results, hepatocytes from fasted rats show a selective twofold decrease in apoBS secretion. Fasting did not affect the relative abundance of apoB RNA, determined by slot blot hybridization assays using two different 32P-labeled cDNA probes coding either for both molecular weight forms or for only the large molecular weight form. However, quantitative of the editing of apoB RNA showed that fasting caused a 60% decrease in the amount of apoB RNA possessing the stop codon. These data show that the editing of apoB RNA is sensitive to metabolic state (i.e., fasting) resulting in a selective decrease in the secretion of apoBS. However, since the total secretion of apoB was decreased by fasting, while apoB mRNA levels remained constant, additional (post-transcriptional) mechanisms play a role in regulating apoB secretion.

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.

Characterization of single base substitutions In edited apolipoprotein B transcripts

Mature RNA transcripts from a single eukaryotic gene may contain different nucleotide sequences, ranging from alternately spliced exons to transcripts from separate alleles differing by only one base. Our laboratory and others have recently reported another class of RNA sequence differences, occurring in transcripts from the single copy apolipoprotein B (apoB) gene. A unique RNA editing mechanism allows expression of the CAA glutamine codon encoded by the apoB gene at nucleotide 6666, or terminates translation by the introduction of a premature UAA translational stop codon. In this study, we used the polymerase chain reaction (PCR) to amplify and characterize edited apoB RNA transcripts differing by a single nucleotide. Amplification and sequence analysis from small quantities of total RNA will facilitate the study of RNA editing and transcription in general.

Thyroid hormone modulates the introduction of a stop codon in rat liver apolipoprotein B messenger RNA.

Apolipoprotein B (apoB) biosynthesis by rat liver was studied following thyroid hormone (3,5,3'-triiodo-L-thyronine) administration to hypothyroid rats. Pharmacologic doses of 3,5,3'-triiodo-L-thyronine caused suppression of apoB100 synthesis but did not affect apoB48 levels. There was no detectable apoB100 synthesis in hyperthyroid rats. To examine whether these results were mediated by the previously demonstrated mechanism of RNA modification (Powell, L. M., Wallis, S. C., Pease, R. J., Edwards, Y. H., Knott, T. J., and Scott, J. (1987) Cell 50, 831-840), the DNA sequence corresponding to the C-terminal end of rat apoB48 was determined from rat liver cDNA clones. Rat cDNAs contained a stop codon at an identical position to that found in human and rabbit apoB48 intestinal cDNA. To quantitate the relative amounts of apoB100 and apoB48 message, cDNA was synthesized from hepatic and intestinal apoB RNA and a 207-base pair fragment amplified using the polymerase chain reaction. The products were then differentially hybridized with oligonucleotides specific for apoB100 (containing CAA) or apoB48 (TAA). Control and hypothyroid liver contained approximately equal amounts of CAA and TAA, while hyperthyroid liver contained greater than 90% TAA. All gut samples contained 94-98% TAA. Genomic DNA from rat liver contained only CAA. The results demonstrate that apoB mRNA modification can be hormonally modulated in the adult rat by induction of a mechanism involving substitution of a stop codon into hepatic apoB100 mRNA.

The human apoB-100 gene: ApoB-100 is encoded by a single copy gene in the human genome

Northern blot analysis of human liver and intestine mRNA revealed two separate apoB mRNA of 14.1 and 7.5 kb in the intestine, and a single 14.1 kb apoB mRNA in the liver. cDNA probes which encode for the 5′, middle, and 3′ regions of the human apoB-100 mRNA have been utilized to evaluate the number of apoB genes present in the human genome by Southern blot hybridization analysis. Comparison of restriction enzyme digestions of high molecular weight leukocyte DNA and a known apoB genomic clone with cDNA probes for the 5′, middle and 3′ regions of the apoB-100 mRNA were consistent with only a single apoB gene per human haploid genome. Further analysis with synthetic oligonucleotides definitively established that there is a single apoB-100 gene. The two mRNA species observed in the intestine must be derived from a single nuclear apoB RNA transcript.