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.

Figure 1. <b>a</b>. Venn diagram showing <i>Apc <sup>min/+</sup></i> -specific 36 APOBEC1-dependent newly identified editing sites. Out of 57 previously identified editing sites in WT C57b/6j intestine, 37 were also edited in both uninvolved mucosa (UM) and/or tumor of <i>Apc <sup>min/+ </sup></i>mice. <b>b</b>. Gross morphology of polyps in jejunum of <i>Apc <sup>min/+</sup></i>, <i>Apc <sup>min/+</sup> Apobec1<sup>–/–</sup></i>, <i>Apc <sup>min/+</sup> Apobec1 <sup>+/Tg</sup></i> and <i>Apc <sup>min/+</sup> RIKO </i>mice.Plyp number and size are shown for each genotype. * p<0.05, *** p< 0.001, **** p<0.0001. <b>c</b>. Top. <i>Flnb</i> RNA editing frequency in individual tumors of 3 separate mice. Bottom. Editing frequency of <i>Flnb</i> RNA in pooled tumors (3-4) isolated from proximal, mid and distal intestine of 7 separate <i>Apc <sup>min/+</sup></i> mice.

Figure 1. a. Venn diagram showing Apc ^min/+ -specific 36 APOBEC1-dependent newly identified editing sites. Out of 57 previously identified editing sites in WT C57b/6j intestine, 37 were also edited in both uninvolved mucosa (UM) and/or tumor of Apc ^min/+mice. b. Gross morphology of polyps in jejunum of Apc ^min/+, Apc ^min/+ Apobec1^–/–, Apc ^min/+ Apobec1 ^+/Tg and Apc ^min/+ RIKO mice.Plyp number and size are shown for each genotype. * p<0.05, *** p< 0.001, **** p<0.0001. c. Top. Flnb RNA editing frequency in individual tumors of 3 separate mice. Bottom. Editing frequency of Flnb RNA in pooled tumors (3-4) isolated from proximal, mid and distal intestine of 7 separate Apc ^min/+ mice.

Figure 2. <b>a</b>. Left, <i>Flnb</i> RNA is significantly downregulated in <i>Apc <sup>min/+</sup></i> <i>RIKO</i> uninvolved and tumor tissue. * p<0.05, *** p<0.001. Middle, FLNB expression is reduced in <i>Apc <sup>min/+</sup> RIKO</i> compared to <i>Apc <sup>min/+</sup></i> mice. ACTIN is used as loading control (n=3 per genotype). Right, RNA decay assay showing reduced <i>Flnb</i> RNA half-life upon loss of RBM47. <b>b</b>. TCGA survey of 524 colorectal adenocarcinoma patients for RBM47 and FLNB showing a subset of patients with both reduced <i>RBM47</i> and <i>FLNB</i> RNA (red square). <b>c</b>. Left, <i>FLNB</i> RNA is significantly downregulated in CRC patients * p<0.05 (n=14 uninvolved-tumors pairs). Right, positive correlation between <i>FLNB</i> and <i>RBM47</i> RNAs in CRC uninvolved and tumor tissues, by Q-PCR analysis.

Figure 2. a. Left, Flnb RNA is significantly downregulated in Apc ^min/+ RIKO uninvolved and tumor tissue. * p<0.05, *** p<0.001. Middle, FLNB expression is reduced in Apc ^min/+ RIKO compared to Apc ^min/+ mice. ACTIN is used as loading control (n=3 per genotype). Right, RNA decay assay showing reduced Flnb RNA half-life upon loss of RBM47. b. TCGA survey of 524 colorectal adenocarcinoma patients for RBM47 and FLNB showing a subset of patients with both reduced RBM47 and FLNB RNA (red square). c. Left, FLNB RNA is significantly downregulated in CRC patients * p<0.05 (n=14 uninvolved-tumors pairs). Right, positive correlation between FLNB and RBM47 RNAs in CRC uninvolved and tumor tissues, by Q-PCR analysis.

Faisal Shurafa

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

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