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.     

Phylogenetic analysis of the apolipoprotein B mRNA-editing region. Evidence for a secondary structure between the mooring sequence and the 3' efficiency element

Apolipoprotein (apo) B mRNA editing is the deamination of C(6666) to uridine, which changes the codon at position 2153 from a genomically encoded glutamine (CAA) to an in-frame stop codon (UAA). The apoB mRNA-editing enzyme complex recognizes the editing region of the apoB pre-mRNA with exquisite precision. Four sequence elements spanning 139 nucleotides (nt) on the apoB mRNA have been identified that specify this precision. In cooperation with the indispensable mooring sequence and spacer element, a 5' efficiency element and a 3' efficiency element enhance editing in vitro. A phylogenetic comparison of 32 species showed minor differences in the apoB mRNA sequence, and the apoB mRNA from 31 species was robustly edited in vitro. However, guinea pig mRNA was poorly edited. Compared with the consensus sequences of these 31 species, guinea pig apoB mRNA has three variations in the 3' efficiency element, and the conversion of these to the consensus sequence increased editing to the levels in the other species. From this information, a model for the secondary structure was formulated in which the mooring sequence and the 3' efficiency element form a double-stranded stem. Thirty-one mammalian apoB mRNA sequences are predicted to form this stem positioning C(6666) two nucleotides upstream of the stem. However, the guinea pig apoB mRNA has a mutation in the 3' efficiency element (C(6743) to U) that predicts an extension of the stem and hence the lower editing efficiency. A test of this model demonstrated that a single substitution at 6743 (U to C) in the guinea pig apoB mRNA, that should reduce the stem, enhanced editing, and mutations in the 3' efficiency element that extended the stem for three base pairs dramatically reduced editing. Furthermore, the addition of a 20-nucleotide 3' efficiency element RNA, to a 58-nucleotide guinea pig apoB mRNA lacking the 3' efficiency element more than doubled the in vitro editing activity. Based on these results, a model is proposed in which the mooring sequence and the 3' efficiency element form a double-stranded stem, thus suggesting a mechanism of how the 3' efficiency element enhances editing.     

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.     

An additional editing site is present in apolipoprotein B mRNA.

Human intestinal apolipoprotein (apo) B mRNA undergoes a C to U RNA editing at nucleotide 6666 to generate a translation stop at codon 2153, which defines the carboxy-terminal of apo B48. Here we show that two of eleven human intestinal cDNAs spanning residue 6666 were edited from a genomically-encoded C to a T at residue 6802 as well as at residue 6666. This additional editing converts Thr (ACA) codon 2198 to Ile (AUA). Synthetic RNA including the nucleotide 6802 was edited in vitro by intestinal extracts at 10-15% of the editing efficiency of nucleotide 6666. A sequence is identified as important for recognition by the editing activity. No secondary structural homology was identified between the two edited sites. No other sequence in the region between 6411 and 6893 nucleotides of apo B mRNA was found to be edited in vivo or in vitro. Apo B RNA editing extracts from intestine did not edit maize cytochrome oxidase II mRNA.     

NMR structure of the apoB mRNA stem-loop and its interaction with the C to U editing APOBEC1 complementary factor

We have solved the NMR structure of the 31-nucleotide (nt) apoB mRNA stem-loop, a substrate of the cytidine deaminase APOBEC1. We found that the edited base located at the 5' end of the octa-loop is stacked between two adenosines in both the unedited (cytidine 6666) and the edited (uridine 6666) forms and that the rest of the loop is unstructured. The 11-nt "mooring" sequence essential for editing is partially flexible although it is mostly in the stem of the RNA. The octa-loop and the internal loop in the middle of the stem confer this flexibility. These findings shed light on why APOBEC1 alone cannot edit efficiently the cytidine 6666 under physiological conditions, the editing base being buried in the loop and not directly accessible. We also show that APOBEC1 does not specifically bind apoB mRNA and requires the auxiliary factor, APOBEC1 complementary factor (ACF), to edit specifically cytidine 6666. The binding of ACF to both the mooring sequence and APOBEC1 explains the specificity of the reaction. Our NMR study lead us to propose a mechanism in which ACF recognizes first the flexible nucleotides of the mooring sequence (the internal loop and the 3' end octa-loop) and subsequently melts the stem-loop, exposing the amino group of the cytidine 6666 to APOBEC1. Thus, the flexibility of the mooring sequence plays a central role in the RNA recognition by ACF.     

Apolipoprotein B RNA sequence 3' of the mooring sequence and cellular sources of auxiliary factors determine the location and extent of promiscuous editing.

Apolipoprotein B (apoB) RNA editing involves a cytidine to uridine transition at nucleotide 6666 (C6666) 5' of an essential cis -acting 11 nucleotide motif known as the mooring sequence. APOBEC-1 (apoB editing catalytic sub-unit 1) serves as the site-specific cytidine deaminase in the context of a multiprotein assembly, the editosome. Experimental over-expression of APOBEC-1 resulted in an increased proportion of apoB mRNAs edited at C6666, as well as editing of sites that would otherwise not be recognized (promiscuous editing). In the rat hepatoma McArdle cell line, these sites occurred predominantly 5' of the mooring sequence on either rat or human apoB mRNA expressed from transfected cDNA. In comparison, over-expression of APOBEC-1 in HepG2 (HepG2-APOBEC) human hepatoma cells, induced promiscuous editing primarily 5' of the mooring sequence, but sites 3' of the C6666 were also used more efficiently. The capacity for promiscuous editing was common to rat, rabbit and human sources of APOBEC-1. The data suggested that differences in the distribution of promiscuous editing sites and in the efficiency of their utilization may reflect cell-type-specific differences in auxiliary proteins. Deletion of the mooring sequence abolished editing at the wild type site and markedly reduced, but did not eliminate, promiscuous editing. In contrast, deletion of a pair of tandem UGAU motifs 3' of the mooring sequence in human apoB mRNA selectively reduced promiscuous editing, leaving the efficiency of editing at the wild type site essentially unaffected. ApoB RNA constructs and naturally occurring mRNAs such as NAT-1 (novel APOBEC-1 target-1) that lack this downstream element were not promiscuously edited in McArdle or HepG2 cells. These findings underscore the importance of RNA sequences and the cellular context of auxiliary factors in regulating editing site utilization.     

Three distinct RNA sequence elements are required for efficient apolipoprotein B (apoB) RNA editing in vitro.

Apolipoprotein B (apoB) mRNA is edited in rat liver and intestine to convert a CAA glutamine codon to a UAA translational stop codon by the direct conversion of cytidine to uridine at nucleotide 6666. We have proposed the 'mooring sequence' model for apoB RNA editing, in which editing complexes (editosomes) assemble on specific apoB mRNA flanking sequences to direct this site-specific editing event. One sequence element (approx. nts 6671-81, the presumed 'mooring sequence') has been previously identified as necessary for editing. We have identified two additional sequence elements which are necessary for efficient editing: (1) a 5' 'Regulator' region which modulates editing efficiency and (2) a 'Spacer' region between the editing site and the 3' mooring sequence, whose distance is critical for efficient editing. Utilizing this data, we have induced editing at a cryptic site and have defined a 22 nucleotide 'cassette' of specific apoB sequence which is sufficient to support wild-type levels of editing in vitro in a background of distal apoB RNA sequence.     

Elimination of apolipoprotein B48 formation in rat hepatoma cell lines transfected with mutant human apolipoprotein B cDNA constructs.

Rat hepatoma McA-RH7777 cell lines transfected with full-length human apolipoprotein (apo) B constructs produce mostly human apoB48 and only small amounts of apoB100, as a result of mRNA editing at codon 2153 (C to U conversion at nucleotide 6666). To abolish the formation of apoB48 and increase the yield of apoB100 and other forms of apoB longer than apoB48, site-specific mutations were introduced at or near the site of apoB mRNA editing. Among four mutations examined, only that in which codon 2153 was converted from CAA (Gln) to CTA (Leu) effectively precluded the formation of apoB48. In this mutant, a stop codon would not be generated even if the C to U conversion occurred. The three other mutations were introduced to disrupt the proposed stem-loop structure encompassing the editing site. Changes made in the third positions of five codons on the 5' side of the edited base or of four codons 3' of the edited base failed to eliminate the production of a protein with the approximate size of apoB48. A construct in which codon 2153 was changed from CAA to GAT (Asp) also failed to eliminate the production of a protein the size of apoB48. Analysis of the region between nucleotides 6200 and 6900 of the cDNA did not detect any prevalent alternate editing sites. Immunoblot analysis using polyclonal antibodies raised against synthetic peptides of human apoB100 indicated that the carboxyl terminus of the apoB48-like proteins probably resides between amino acid residues 2068 and 2129 of apoB100. These results provide some insight into the mechanism of apoB mRNA editing and will facilitate further studies on apoB-containing lipoproteins.     

Apolipoprotein B mRNA sequences 3'' of the editing site are necessary and sufficient for editing and editosome assembly.

Apolipoprotein B (apoB) mRNA is edited in rat liver and intestine through the direct conversion of cytidine to uridine at nucleotide 6666. Recently, we have proposed the 'Mooring Sequence' model, in which editing complexes (editosomes) assemble on specific apoB mRNA flanking sequences to direct this site-specific editing event. To test this model, apoB mRNA deletion and translocation mutants were constructed and analyzed. Specific sequences 3' of the editing site were absolutely required for editing, while specific sequences and bulk RNA 5' of the editing site were required for efficient editing. Translocation of apoB 3' flanking sequences induced editing of an upstream cytidine, demonstrating that 3' sequences are necessary and sufficient to direct editing in vitro. 3' flanking sequences were also shown to be necessary and sufficient for editosome complex assembly. These data provide strong support for a 'Mooring Sequence' model in which 3' apoB flanking sequences direct editosome assembly and subsequent editing in vitro, while 5' flanking sequences enhance these functions.     

Partially edited RNAs are intermediates of RNA editing in plant mitochondria

RNA editing in flowering plant mitochondria addresses several hundred specific C nucleotides in individual sequence contexts in mRNAs and tRNAs. Many of the in vivo steady state RNAs are edited at some sites but not at others. It is still unclear whether such incompletely edited RNAs can either be completed or are aborted. To learn more about the dynamics of the substrate recognition process, we investigated in vitro RNA editing at a locus in the atp4 mRNA where three editing sites are clustered within four nucleotides. A single cis-element of about 20 nucleotides serves in the recognition of at least two sites. Competition with this sequence element suppresses in vitro editing. Surprisingly, unedited and edited competitors are equally effective. Experiments with partially pre-edited substrates indicate that indeed the editing status of a substrate RNA does not affect the binding affinity of the specificity factor(s). RNA molecules in which all editing sites are substituted by either A or G still compete, confirming that editing site recognition can occur independently of the actual editing site. These results show that incompletely edited mRNAs can be substrates for further rounds of RNA editing, resolving a long debated question.