The Neurofibromatosis Type I Messenger RNA
Undergoes Base-Modification RNA Editing

Gary R. Skuse, Amedeo J. Cappione, Mark Sowden

Linda J. Metheny and Harold C. Smith


ABSTRACT: A functional mooring sequence, known to be required for apolipoprotein B (apoB) mRNA editing, exists in the mRNA encoding the neurofibromatosis type I (NF1) tumor suppressor. Editing of NF1 mRNA modifies cytidine in an arginine codon (CGA) at nucleotide 2914 to a uridine (UGA), creating an in frame translation stop codon. NF1 editing occurs in normal tissue but was several-fold higher in tumors. In vitro editing and transfection assays demonstrated that apoB and NF1 RNA editing will take place in both neural tumor and hepatoma cells. Unlike apoB, NF1 editing did not demonstrate
dependence on rate-limiting quantities of APOBEC-1 (the apoB editing catalytic subunit) suggesting that different trans-acting factors may be involved in the two editing processes.

INTRODUCTION: RNA editing involves specific alterations of genetic information at the transcriptional or post-transcriptional levels. This process has been described in diverse eukaryotic organisms and can be mechanistically categorized as either RNA strand scission and ligation (insertion editing) or base-modification editing. There are four currentexamples of mammalian cellular RNA editing. Each belongs to the base modification category of editing yet no common mechanism has emerged.

Apolipoprotein B (apoB) mRNA editing involves cytidine to uridine conversion at nucleotide 6666 and creates an in-frame translation stop codon (UAA) from a glutamine codon (CAA). Proteins translated from both edited and unedited apoB mRNAs participate in lipid binding and transport to peripheral body tissue. Lipoprotein particles assembled on the truncated protein are cleared from the blood more rapidly and hence present a reduced risk factor in the acquisition of atherogenic diseases. Site-specific editing of apoB mRNA is mediated by a cytidine deaminase, APOBEC-1whose activity on RNA is dependent upon its assembly with one or more auxiliary proteins as an editosome.

Site-directed mutagenesis has defined the apoB mRNA editing site. It consists of a tripartite, 21 nucleotide motif containing a spacer sequence element and a mooring sequence, both 3' of the cytidine to be edited, and a regulator element immediately 5' of the cytidine. The mooring sequence is the only element within the tripartite motif which is necessary and sufficient for site-specific editing. Recognition of the mooring sequence during editosome assembly has been largely attributed to RNA-binding proteins of 40 to 66 kDa, although APOBEC-1 may have a non-specific and low affinity RNA-binding capacity.

Editing involving adenosine deamination has been demonstrated in mRNA encoding receptor subunits (GluR-B) of the AMPA subtype of glutamate-gated ion channels which mediate fast excitatory neurotransmission. Adenosine is deaminated to form inosine in the context of a CAG glutamine (Q) codon. The resultant CIG is thought to be translated as a CGG arginine (R) codon. The Q/R amino acid substitution occurs within the second hydrophobic segment of the protein and is essential for reducing Ca2+ permeability of ion channels containing the GluR-B subunit. Emerging details of the mechanism for site-specific editing suggest that editing site recognition is mediated through RNA secondary structure formed between unique RNA sequences flanking the editing site in exon 11 and an exon-complementary sequence (ECS) within the downstream intron. A double-stranded RNA adenosine deaminase, believed to be responsible for GluR-B mRNA editing, has been isolated from mammalian tissues and characterized.

Editing of the Wilms' tumor suppressor gene product (WT1) mRNA has been observed in both human and rat tissues and appears to involve amidation of uridine to cytidine at nucleotide position 839. Edited WT1 (containing a proline (CCC) substituted for leucine (CUC)) was less effective in regulating transcription in transfected cells from the early growth responsepromoter, suggesting a potential role for editing in the pathogenesis of Wilms' tumor.

In addition to mRNA editing, tRNAAsp editing has been described in rodents and marsupials. Editing of tRNAAsp in both species has been proposed to be essential as the modified tRNAs participate in translation. The majority of rat liver cytoplasmic tRNAAsp is edited at two sites adjacent to the anticodon loop involving a CĂU and UĂC modification at positions 32 and 33 respectively. Up to 50% of marsupial mitochondrial tRNAAsp is also edited but modification occurs in this instance within the anticodon and involves a GCC to GUC conversion. The mechanism(s) and factors remain to be determined in what appears to be both deamination and amidation processes.

The number of examples of RNA editing in mammals and the diversity of the biological systems affected suggest that editing may be a common mechanism for regulating gene expression. We have evaluated the occurrence of mooring sequences within known mRNAs through the aid of computer data base searches. An editing site within the mRNA encoding the neurofibromatosis type I (NF1) gene product, neurofibromin, has been identified which is predicted to result in loss of tumorsuppressor gene product function due to the introduction of a premature, in-frame translation stop codon.

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