The histone variant H2A.Z promotes efficient co-transcriptional splicing in S. cerevisiae

In eukaryotes, a dynamic ribonucleic protein machine known as the spliceosome catalyzes the removal of introns from pre-messenger RNA (pre-mRNA). Recent studies show the process of RNA-synthesis and RNA-processing to be spatio-temporally coordinated, indicating that RNA splicing takes place in the context of chromatin. H2A.Z is a highly conserved histone variant of the canonical histone H2A. In S. cerevisiae, H2A.Z is deposited into chromatin by the SWR1-complex, is found near the 5’ ends of protein-coding genes, and has been implicated in transcription regulation. Here we show that splicing of intron-containing genes in cells lacking H2A.Z is impaired, particularly under suboptimal splicing conditions. Cells lacking H2A.Z are especially dependent on a functional U2 snRNP, as H2A.Z shows extensive genetic interactions with U2 snRNP associated proteins, and RNA-seq reveals introns with non-consensus branch points are particularly sensitive to H2A.Z loss. Consistently, H2A.Z promotes efficient spliceosomal rearrangements involving the U2 snRNP, as H2A.Z loss results in persistent U2 snRNP association and decreased recruitment of downstream snRNPs to nascent RNA. H2A.Z impairs transcription elongation, suggesting that spliceosome rearrangements are tied to H2A.Z’s role in elongation. Depletion of disassembly factor Prp43 suppresses H2A.Z-mediated splice defects, indicating that, in the absence of H2A.Z, stalled spliceosomes are disassembled and unspliced RNAs are released. Together these data demonstrate that H2A.Z is required for efficient pre-mRNA splicing and indicate a role for H2A.Z in coordinating the kinetics of transcription elongation and splicing.在真核生物中，一类被称为剪接体（spliceosome）的动态核糖核蛋白复合物，可催化从前信使RNA（pre-messenger RNA，pre-mRNA）中切除内含子。近期研究表明，RNA合成与RNA加工过程存在时空协同性，这提示RNA剪接是在染色质（chromatin）环境中进行的。H2A.Z是经典组蛋白H2A的一种高度保守的组蛋白变体。在酿酒酵母（S. cerevisiae）中，H2A.Z由SWR1复合物（SWR1-complex）沉积到染色质中，定位于蛋白质编码基因的5'端附近，并被证实参与转录调控。本研究发现，缺失H2A.Z的细胞中，含内含子基因的剪接过程会受损，在非最优剪接条件下这一缺陷尤为显著。缺失H2A.Z的细胞对功能性U2小核糖核蛋白颗粒（U2 snRNP）具有高度依赖性——这是因为H2A.Z与U2 snRNP结合蛋白存在广泛的遗传互作，且RNA测序（RNA-seq）结果显示，带有非共识型分支点的内含子对H2A.Z缺失尤为敏感。与此一致的是，H2A.Z可促进涉及U2 snRNP的高效剪接体重排：H2A.Z缺失会导致U2 snRNP持续结合，并降低下游小核糖核蛋白颗粒向新生RNA的招募效率。H2A.Z会抑制转录延伸过程，这提示剪接体重排与H2A.Z在转录延伸中的功能存在关联。敲低剪接体解离因子Prp43可缓解H2A.Z介导的剪接缺陷，这表明在缺失H2A.Z的情况下，停滞的剪接体会被解离并释放未剪接的RNA。综上，本研究数据证实H2A.Z对于高效的前信使RNA剪接是必需的，并揭示了H2A.Z在协调转录延伸与剪接的动力学过程中发挥的作用。