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

Abstract

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.

Apolipoprotein B mRNA and lipoprotein secretion are increased in McArdle RH-7777 cells by expression of betaine-homocysteine S-methyltransferase

“…Cytidine at nucleotide 6666 (Cyt-6666) is deaminated to uridine, thereby altering a glutamine codon to a stop codon, resulting in the translation of a protein that is only 48 % of the full-length apo B. This process is mediated by the editosome, a multiprotein complex [22]. Since a fraction containing this complex was used as the antigen preparation for the generation of the monoclonal antibodies, we determined whether our cell lines expressing BHMT exhibited altered apo B-mRNA-editing efficiencies.…”

Absence of APOBEC-1 mediated mRNA editing in human carcinomas

“…The editing of apo B mRNA is mediated by a multicomponent enzyme-complex, designated apo B mRNA editing enzyme Smith et al, 1991). The catalytic subunit APOBEC-1 (apo B mRNA editing enzyme catalytic polypeptide 1) is the only component of this enzyme-complex that has been conclusively characterized .…”

Commitment of apolipoprotein B RNA to the splicing pathway regulates cytidine-to-uridine editing-site utilization

“…An explanation for this is that, despite the fact that the apoB editing site resides in the centre of the 7.5 kb exon 26, editosome assembly and\or function may be inhibited by spliceosome assembly and\or function. In this regard, 500-2000 nt of apoB RNA expressed from cDNA edited with a 2-fold or greater efficiency in McArdle rat hepatoma cells than the natively expressed apoB mRNA [10,11]. Cytidine-to-uridine editing required only a 21 nt tripartite motif surrounding the edited cytidine residue.…”

“…This motif includes an 11-nucleotide mooring sequence 3h of C'''', a 4 nt spacer element between the edited cytidine and the mooring sequence and an enhancer element immediately upstream of C'''' [12][13][14]. Though less efficient, transcripts as short as 55 nt supported editing [11,15,16], and the tripartite motif alone supported editing when placed in a foreign RNA context [12,17]. Subsequently, additional sequences 5h and 3h of the tripartite motif were shown to contain efficiency elements [15,16,18,19] and secondary structure [13,14,20,21] that promoted editosome assembly and editing activity.…”

“…Editosome assembly in cells [22,26] or in itro [11,15,22,27] required a complex protein composition with an aggregate size of 27 S. Minimally, in itro editing activity required a homodimer of APOBEC-1 (catalytic subunit for cytidine-to-uridine editing of apolipoprotein B mRNA-1), the zinc-dependent catalytic subunit of cytidine deaminase [28,29] and a 65 kDa RNA binding protein, ACF\ASP (APOBEC-1 complementation factor\APOBEC-1 stimulatory protein) [30,31]. APOBEC-1 demonstrated a weak and non-specific RNA binding activity to AU-rich apoB sequences [32] and alone could not edit apoB mRNA.…”