RBM47 REGULATES FUNCTIONAL EXPRESSION OF NOVEL C-TO-U RNA EDITING TARGETS AND TUMORIGENESIS IN MOUSE AND HUMAN INTESTINE

Background: C-to-U RNA editing is mediated by APOBEC1 and two RNA binding proteins (A1CF and RBM47) in WT C57 mice. We found reduced polyp burden in Apc^min/+ mice crossed into Apobec1^–/– mice, whose mechanisms are unknown. Here we explored the relative importance of epithelial APOBEC1 and RBM47 expression and identified novel RNA targets and mechanisms of regulation. Approach: Apc^min/+ mice were crossed into Rbm47 intestinal knockout (Apc-RIKO) and intestinal human Apobec1 transgenic mice (Apc-A1tg) to examine gain- and loss-of-function (GOF/LOF) of C-to-U RNA editing. RNA from Apc^min/+ polyp and uninvolved mucosa (UM) was pooled from >5 mice, deep sequenced and variants Sanger-sequenced to verify C-to-U RNA editing. Validated targets were examined in patients with familial adenomatous polyposis (FAP) or sporadic colorectal cancer (CRC). Findings: 36 novel, APOBEC1-dependent editing sites were identified in the 3’UTR of 29 RNAs in Apc^min/+ mice. 6 were edited only in polyp, 12 only in UM and 18 in both UM and polyp (Fig1a); 37 of 57 editing sites previously identified in C57 WT mice were validated in Apc ^min/+ mice. An RNA sequence composition /folding prediction model examining shared C57+Apc ^min/+ RNAs revealed that editing efficiency correlated with the AU content of Apc ^min/+ but not C57-specific targets. 23/29 APOBEC1 RNA targets were RBM47-dependent. QPCR analysis of Apobec1, Rbm47 and A1cf RNAs in C57 vs Apc ^min/+ mice showed reduced Rbm47 and increased A1cf in Apc^min/+ polyp, suggesting that editing efficiency is regulated via APOBEC1/RBM47/A1CF stoichiometry. GOF Apc-A1tg mice showed increased polyp burden and size (Fig1b) with only 21/36 sites edited (6 higher frequency, 4 lower frequency and 11 similar frequency vs Apc^min/+ mice); LOF Apc -RIKO mice showed larger polyps but similar overall burden (Fig1b), again suggesting a stoichiometric requirement for APOBEC1/RBM47. 3/29 RNAs showed altered expression in LOF Apc ^min/+ Apobec1^–/– and Apc-RIKO (Flnb, Herc6, Casp8ap2). Flnb RNA (95 % edited in UM and polyps) was >50% edited in individual tumors while individual mice showed regional variability in proximal and mid intestine but consistently ~90% editing in distal region (Fig1c). We observed reduced Flnb mRNA and protein in Apc-RIKO mice (Fig2a) with decreased Flnb RNA half-life suggesting that loss of RBM47 promotes tumor growth via altering Flnb RNA stability (Fig2a). TCGA of 524 colorectal adenocarcinoma patients revealed a subset with reduced RBM47 and FLNB RNAs (Fig2b). FNLB mRNA was also decreased in CRC samples, correlating with RBM47 RNA expression (Fig2c). Conclusions: We identified 36 novel C-to-U RNA editing sites across 29 targets in Apc ^min/+ mice that exhibit potential roles in intestinal polyposis in mouse and human tissues with cofactor (RBM47) dependence influencing altered mRNA stability and tumorigenesis.