Caffeine modulates post-transcriptional regulation of SRSF 2 1 Methylxanthines Increase Expression of the Splicing Factor SRSF 2 by Regulating Multiple Post-Transcriptional Mechanisms

We have previously reported that the methylxanthine caffeine increases expression of the splicing factor SRSF2, the levels of which are normally controlled by a negative auto-regulatory loop. In the present study we have investigated the mechanism by which methylxanthines induce this aberrant overexpression. RT-PCR analyses suggested little impact of caffeine on SRSF2 total mRNA levels. Instead, caffeine induced changes in the levels of SRSF2 3ʹUTR splice variants. While some of these variant were substrates for nonsense-medicated decay (NMD), and could potentially have been stabilized by caffeine-mediated inhibition of NMD, downregulation of NMD by a genetic approach was not sufficient to reproduce the phenotype. Furthermore, cell-based assays demonstrated that some of the caffeineinduced variants were intrinsically more efficiently translated than others; the addition of caffeine increased the translational efficiency of most SRSF2 transcripts. MicroRNA array analyses revealed a significant caffeine-mediated decrease in the expression of two SRSF2targeting miRs, both of which were shown to repress translation of specific SRSF2 splice variants. These data support a complex model whereby caffeine downregulates SRSF2-targeting microRNAs, leading to an increase in SRSF2 translation, which in turn induces SRSF2 splicing. SRSF2 splice variants are then stabilized by caffeinemediated NMD inhibition, breaking the normal negative feedback loop and allowing the aberrant increase in SRSF2 protein levels. These findings highlight the complexity of SRSF2 gene regulation, and suggest ways in which SRSF2 expression may be dysregulated