Apobec1 complementation factor (A1CF) and RBM47 interact in tissue-specific regulation of C to U RNA editing in mouse intestine and liver

Abstract

Mammalian C to U RNA is mediated by APOBEC1, the catalytic deaminase, together with RNA binding cofactors (including A1CF and RBM47) whose relative physiological requirements are unresolved. Although A1CF complements APOBEC1 for in vitro RNA editing, A1cf -/mice exhibited no change in apolipoproteinB (apoB) RNA editing, while Rbm47 mutant mice exhibited impaired intestinal RNA editing of apoB as well as other targets. Here we examined the role of A1CF and RBM47 in adult mouse liver and intestine, following deletion of either one or both gene products and also following forced (liver or intestinal) transgenic A1CF expression. There were minimal changes in hepatic and intestinal apoB RNA editing in A1cf -/mice and no changes in either liver-or intestine-specific A1CF transgenic mice. Rbm47 liver-specific knockout (Rbm47 LKO ) mice demonstrated reduced editing in a subset (11 of 20) of RNA targets, including apoB. By contrast, apoB RNA editing was virtually eliminated (<6% activity) in intestine-specific (Rbm47 IKO ) mice with only five of 53 targets exhibiting C-to-U RNA editing. Double knockout of A1cf and Rbm47 in liver (AR LKO ) eliminated apoB RNA editing and reduced editing in the majority of other targets, with no changes following adenoviral APOBEC1 administration. Intestinal double knockout mice (AR IKO ) demonstrated further reduced editing (<10% activity) in four of five of the residual APOBEC1 targets identified in AR IKO mice. These data suggest that A1CF and RBM47 each function independently, yet interact in a tissue-specific manner, to regulate the activity and site selection of APOBEC1 dependent C-to-U RNA editing.

Comparison of RNA Editing Activity of APOBEC1-A1CF and APOBEC1-RBM47 Complexes Reconstituted in HEK293T Cells

“…It is clear that human A1CF can mediate APO1 editing activity on APOB RNA in this cellbased assay system, but its effectiveness on activity is about 73% of RBM47; this trend is consistent with the relative impact that knockout of mouse A1cf and Rbm47 has on Apob editing activity in vivo as was recently reported [27]. This same trend was observed when testing the mouse homologs of these proteins with this cell-based assay system, although mA1CF was observed to be slightly weaker for aiding APOB editing activity: mA1CF is only about 52% as effective at inducing editing as mRBM47.…”

“…For the most part, this data correlates well with what has been suggested by mouse in vivo studies on substrate specificity of the two cofactors [27], and many of the substrates shown to be favored by RBM47 in that past work were similarly favored here. However, a few key differences stand out.…”

“…It is notable that RBM47 was so clearly favored over A1CF in terms of editing activity of APOB RNA, as this aligns well with previous reports suggesting that A1CF may not be the favored cofactor for Apob RNA editing under normal physiological conditions [21,22]. It is also very consistent with the most recent report that revealed involvement of both RBM47 and A1CF on Apob RNA editing in mouse hepatic cells [27], with RBM47 having a greater impact than A1CF on this editing activity. This consistency with in vivo methods provides excellent validation of the methodology.…”

“…Another key difference was that editing of endogenous Sh3bgrl mRNA was preferentially affected by the mouse A1cf knockout in vivo [27], whereas the Sh3bgrl substrate showed no statistically significant levels of editing in the presence of mouse mAPO1 and mA1CF with this assay. This may be due to differences in the system: it is possible the human-derived HEK293T cells failed to make a key post-translational modification the mouse proteins needed, or perhaps some other regulatory cofactor is missing.…”

“…It was our goal to better characterize the domains of RBM47 needed for complementation of APO1 editing activity, and to compare the relative editing activity of RBM47 and A1CF in complex with APO1 on both APOB and several other described RNA targets. During the preparation of this manuscript, a new report demonstrated the tissue-specific regulation of APO1 activity by A1CF and RBM47 on multiple endogenous RNA targets through the use of knockout mouse models [27]. We report here a methodology that allows for direct assessment of RNA editing activity in cells using a novel fluorescence-based assay.…”