RNA-Mediated Regulation of Concentrative Nucleoside Transporter 3

Abstract ID 128607 Poster Board 252 Background: Single nucleotide polymorphisms in the SLC28A3 locus (encoding Concentrative Nucleoside Transporter 3 (CNT3)) have been shown to associate with reduced doxorubicin-induced cardiotoxicity. These include a synonymous genomic variant rs7853758 (G > A, L461L) and a variant in its antisense long noncoding RNA (lncRNA, AS1) rs11140490. However, there is lack of knowledge on the mechanistic action of these variants and AS1 in regulating CNT3. We hypothesized that these genomic variants and AS1 play essential roles by altering CNT3 mRNA splicing and/or transcript levels, and therefore its expression. Methods: Three minigene constructs (ex14-15, ex13-14-15 partial, and ex13-14-15 full) containing the WT and SNP rs7853758 in exon 14 of SLC28A3 were generated and subjected to mRNA splicing and real time RT-PCR analyses in HEK293 cells. Minigene constructs were also created containing intronic regions and all of exons 1 and 2 of SLC28A3 AS1 lncRNA and subjected to splicing and real time RT-PCR assays. AS1 expression constructs were generated from endogenously spliced WT AS1, AS1 containing a single-nucleotide change (generated by site-directed mutagenesis), and two other endogenously spliced variants of AS1 with deletions in exon 2 and at the 3' end. The latter constructs were evaluated for their effects on CNT3 transcript and protein levels using RT-PCR and Western blotting assays. Results: Minigene splicing assays demonstrated proper splicing of SLC28A3 with the ex14-15 and ex13-14-15 full constructs at various time points. However, the ex13-14-15 partial construct showed reduced levels of spliced SLC28A3 transcripts, suggesting that the regulatory elements required for splicing are located deep within int13. SNP rs7853758 did not result in an altered splicing pattern, however it resulted in decreased SLC28A3 transcripts. AS1 lncRNA minigene splicing assay detected the successful generation of the mature AS1 transcript in HEK293 cells (designated as AS-1 original). Surprisingly, the assay also detected two other novel forms of AS1 with deletions in exon 2 and towards the 3' end designated as AS-1-deletion and AS-1-trimmed, respectively. However, the presence of rs11140490 SNP on the exon/intron boundary of AS1 precursor did not result in visual differences in splicing pattern or in AS1 mature transcript levels. Increased expression of AS1 isoforms resulted in the reduction of CNT3 transcripts as measured from RT-PCR analysis of full-length and AS1 binding region of CNT3 transcripts. The decrease in CNT3 transcript levels occurred in a dose-dependent manner with a potency order of AS1 original>AS1 trimmed>AS1 deleted. The reduction in the CNT3 transcripts by AS1 concurred with the reduction in CNT3 protein levels as measured by Western blotting analysis of FLAG-CNT3 in AS1 co-transfected HEK293 cells. When compared with the effects of WT AS1, no significant differences in the expression of CNT3 mRNA or protein levels were observed with SNP introduced AS1. Conclusion: Intron 13 of SLC28A3 contains splicing regulatory elements deep within the intron. AS1 minigene precursors can splice in vivo to generate 3 functional isoforms. Synonymous genomic variant rs7853758 of SLC28A3 and its antisense lncRNA regulate CNT3 transcript and protein expression. These findings implicate that splicing and antisense mechanisms may be utilized to modulate CNT3 function for reducing doxorubicin-induced cardiotoxicity. Funding: Dialysis Clinic Inc.-SA* and OSUCCC-RG*; *Corresponding authors