Table_1_A genetic screen in Arabidopsis reveals the identical roles for RBP45d and PRP39a in 5’ cryptic splice site selection.xlsx

Cryptic splice sites in eukaryotic genome are generally dormant unless activated by mutation of authentic splice sites or related splicing factors. How cryptic splice sites are used remains unclear 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 SOT5 intron 7. Loss-of-function mutations in RBP45d and PRP39a significantly increase the level of a cryptically spliced variant that encodes a mutated but functional sot5 protein, rescuing sot5 to the WT phenotype. We furtherly demonstrated that RBP45d and PRP39a interact with each other and also with the U1C, a core subunit of U1 snRNP. We found that RBP45d directly binds to the uridine (U)-rich RNA sequence downstream the 5’ ss of SOT5 intron 7. However, other RBP45/47 members do not function redundantly with RBP45d, at least in regulation of cryptic splicing. Taken together, RBP45d promotes U1 snRNP to recognize the specific 5’ ss via binding to intronic U-rich elements in plants.

真核生物基因组中的隐匿性剪接位点通常处于休眠状态，除非被真实剪接位点或相关剪接因子的突变所激活。在植物中，隐匿性剪接位点的利用机制尚不明确。本研究通过遗传筛选抑制由SOT5内含子7的5'剪接位点（5' ss）的点突变引起的绿色突变体sot5的抑制子，鉴定出两种隐匿性剪接调控因子RBP45d和PRP39a，它们分别是酵母U1辅助蛋白Nam8和Prp39的同源物。RBP45d和PRP39a的功能缺失突变显著增加了编码突变但功能正常的sot5蛋白的隐匿性剪接变体的水平，从而挽救了sot5至野生型表型。进一步研究表明，RBP45d和PRP39a相互作用，并与U1C（U1小核RNA复合物的一个核心亚基）相互作用。我们发现RBP45d直接结合到SOT5内含子7 5' ss下游的尿苷（U）富集的RNA序列。然而，其他RBP45/47成员并不与RBP45d功能冗余，至少在调节隐匿性剪接方面。综合以上发现，RBP45d通过结合植物内含子中的U富集元素，促进U1小核RNA复合物识别特定的5' ss。