RBP45d is required for 5' splice site selection via binding to intronic U-rich elements and interaction with PRP39a in Arabidopsis thaliana

Abstract

A large number of cryptic splice sites in eukaryotic genome are generally dormant unless activated by mutations of authentic splice sites or splicing factors. How cryptic splice sites are used remains unknown in plants. Here, we identified two cryptic splicing regulators, RBP45d and PRP39a that are homologs of yeast U1 auxiliary protein Nam8 and Prp39, respectively, via genetic screening for suppressors of the virescent sot5 mutant, which results from a point mutation at the 5 splice site (5 ss) of intron 7. PCR and DNA sequencing data showed that loss-of-function mutations in RBP45d and PRP39a significantly increase the level of a cryptically spliced mRNA that encodes a mutated but partially functional sot5 protein, rescuing sot5 to the WT phenotype. Yeast two hybrid and bimolecular fluorescence complementation assays demonstrated that RBP45d and PRP39a interact each other and also with the U1C, a core subunit of U1 small nuclear ribonucleoprotein (U1 snRNP). RNA electronic mobility shift assay showed that RBP45d directly binds to the uridine (U)-rich RNA sequence downstream of the cryptic 5 ss. Consistently, our transcriptomic analysis revealed that a set of introns with U-rich sequences are retained in rbp45d. However, we found that other RBP45/47 members do not function redundantly with RBP45d, at least in regulation of cryptic splicing. Collectively, our data suggest that RBP45d is required for 5 ss selection via binding to intronic U-rich elements and PRP39a in plants. One sentence summaryThe Arabidopsis RBP45d interacting with U1C and PRP39a is required for 5 ss selection via binding to intronic U-rich elements.