Tissue-specific differences in the role of RNA 3' of the apolipoprotein B mRNA mooring sequence in editosome assembly.

Site-specific editing of apolipoprotein B (apoB) mRNA by the cytidine deaminase, APOBEC-1 is proposed to require interactions of auxiliary protein(s) with an eleven nucleotide element, the mooring sequence, located 3' of the C --> U editing site. An analysis of the RNA sequence dependence for protein-RNA interactions and editosome assembly in rat liver and the small intestine demonstrated that the mooring sequence was a minimal requirement for these interactions. Sequences 3' of the mooring sequence either interacted with 66 kDa and 44 kDa proteins or enhanced the interactions of these proteins with the mooring sequence. The data also suggested tissue-specific differences in the relative importance of the 3' cis-acting 'enhancer' elements in the efficiency or stability of editosome assembly. We propose that the previously demonstrated differences in apoB mRNA editing efficiency and its regulation in liver and intestine may in part be due to differences in auxiliary protein interactions with apoB mRNA 3' of the mooring sequence.

Only cytidines 5' of the apolipoprotein B mRNA mooring sequence are edited.

A 22 nucleotide 'cassette' containing three distinct sequence elements is required for efficient apolipoprotein B (apoB) RNA editing. One of the elements, the mooring sequence, has been proposed as the apoB RNA-recognition element in binding 66 and 44 kDa proteins during editosome assembly. We show here that editing can only take place on cytidines 5' of the mooring sequence; thereby providing further proof for the proposed orientation-specific assembly of editing factors at the editing site. We also provide direct evidence for the existence of a cryptic mooring sequence immediately downstream of the wild-type editing site (between nucleotides 6688-6710). When a cytidine is introduced 5' of this site (at nucleotide 6687), editing occurs at wild-type (nucleotide 6666) and cryptic sites. The cytidine to be edited is not involved in 66 and 44 kDa protein binding, and therefore, the data suggest that the cryptic mooring sequence binds these factors, and may facilitate factor recruitment to the wild-type editing site. These data may also explain why wild-type levels of editing on chimeric, RNA editing substrates can only be achieved when the 22 nucleotide editing cassette is flanked 3' by apoB RNA sequence.

Specific 3' sequences flanking a minimal apolipoprotein B (apoB) mRNA editing 'cassette' are critical for efficient editing in vitro.

Apolipoprotein B (apoB) mRNA in mammalian intestine undergoes direct conversion of cytidine to uridine at nucleotide 6666, generating a UAA stop codon which defines the carboxyl-terminus of apoB48. We have identified three distinct sequence elements which are required for efficient apoB RNA editing in vitro and have defined a 20 nucleotide 'editing cassette' which will support efficient editing in the context of a variety of AT-rich apoB mRNA backgrounds. An important question remaining to be addressed is whether this cassette can support editing in a GC-rich background characteristic of other mRNAs. We demonstrate that the context into which the editing cassette is inserted may determine the efficiency of editing site utilization. When an editing cassette is placed in the context of human albumin mRNA sequence, editing is reduced 6-fold relative to a construct of similar length in which the cassette is surrounded by apoB mRNA sequence. Chimeric RNA substrates may be efficiently edited, however, when albumin sequence resides only 5' of the editing cassette. The data suggest that the proximal of an AT-rich, apoB-like, mRNA sequence is only required 3' of the editing cassette. These results should prove useful in evaluating the potential for editing on mRNAs where similar cassettes can be found.

Extract-specific heterogeneity in high-order complexes containing apolipoprotein B mRNA editing activity and RNA-binding proteins.

The mechanism for tissue-specific differences in apolipoprotein B (apoB) mRNA editing efficiency is not known. Structural data are presented which demonstrate tissue-specific, quantitative differences in the high order complexes containing apoB mRNA editing activity and RNA-binding proteins. The bulk of rat enterocyte extract editing activity sedimented at 11 S with an additional 5-10% at 60 S. Rat liver extract activity was less abundant and only sedimented at 60 S. Ultraviolet light cross-linking revealed two protein activities of approximately 66 and 44 kDa which specifically associated with apoB RNA substrates and cosedimented with editing activity. Extracts differed in the cross-linking yield of p66 and p44 and kinetically, enterocyte RNA-protein complexes reached maximum abundance more rapidly than those in liver extracts. Both 60 and 11 S forms of the editing activity redistributed to 27 S during in vitro editosome assembly. The redistribution of editing activities was accompanied by a corresponding redistribution of p66/p44 to 27 S. The data demonstrate that p66 and p44 are common to liver and enterocyte 27 S editosome assembly processes and suggest that differences in both the pre-editosomal assembly state of editing factors and their abundance may be mechanistically important for tissue-specific differences in editing efficiency.

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.

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.

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.

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.