Pre-splicing factors associate with UpA-rich RNA and limit flavivirus replication

The RNA of all living organisms and most viruses exhibits pervasive suppression of uracil-adenine (UpA) dinucleotides, yet the underlying molecular mechanisms remain poorly understood. Here, we use RNA affinity purification coupled with mass spectrometry to identify diverse host factors that preferentially bind UpA-high RNA sequences. The identified proteins include the canonical antiviral proteins IFIT1 and OAS1, as well as multiple components of the U1 and U2 small nuclear ribonucleoproteins (snRNPs), core elements of the splicing machinery. These splicing factors display increased affinity for artificially UpA-enriched RNA and the West Nile virus (WNV) 3' UTR, which naturally contains higher frequencies of UpA dinucleotides. The functional relevance of splicing factor interactions was demonstrated by pharmacological inhibition of early splicing events, which partially restored the replicative fitness of synonymously recoded and otherwise attenuated UpA-high mutants of Zika virus and WNV. Together, these findings reveal a novel antiviral role for splicing factors, linking their preferential association with UpA-rich RNA to the restriction of UpA-high viruses. Given the evolutionary conservation of the splicing machinery, our results suggest that splicing factor-mediated recognition may contribute to maintain the universal suppression of UpA dinucleotides in host and viral RNA.