High-resolution mapping of transcribing RNAPII by CRAC in the presence of different Sen1 variants

Pervasive transcription is a widespread phenomenon leading to the production of a plethora of non-coding RNAs (ncRNAs) without apparent function. Pervasive transcription poses a risk that needs to be controlled to prevent the perturbation of gene expression. In yeast, the highly conserved helicase Sen1 restricts pervasive transcription by inducing termination of non-coding transcription. However, the mechanisms underlying the specific function of Sen1 at ncRNAs are poorly understood. Here we identify a motif in an intrinsically disordered region of Sen1 that mimics the phosphorylated carboxy terminal domain (CTD) of RNA polymerase II and characterize structurally its recognition by the CTD-interacting domain of Nrd1, an RNA-binding protein that binds specific sequences in ncRNAs. In addition, we show that Sen1-dependent termination strictly requires the recognition of the Ser5-phosphorylated form of the CTD by the N-terminal domain of Sen1. Furthermore, we find that the N-terminal and the C-terminal domains of Sen1 can mediate intra-molecular interactions. Our results shed light onto the network of protein-protein interactions that control termination of non-coding transcription by Sen1.普遍转录是一种广泛存在的生命现象，可产生大量无明显功能的非编码RNA（ncRNAs）。该过程会带来潜在风险，需通过调控加以规避，以防止基因表达发生紊乱。在酵母中，高度保守的解旋酶Sen1可通过诱导非编码转录终止，抑制异常的普遍转录活动。然而，学界对Sen1在非编码RNA上发挥特异性功能的分子机制仍了解甚少。本研究鉴定出Sen1固有无序区域中的一段功能基序，该基序能够模拟RNA聚合酶II的磷酸化羧基末端结构域（CTD）；同时从结构层面解析了RNA结合蛋白Nrd1的CTD相互作用结构域对该基序的识别过程——Nrd1可特异性结合非编码RNA中的特定序列。此外，本研究证实依赖Sen1的转录终止严格需要Sen1的N末端结构域识别CTD的Ser5磷酸化形式。进一步研究发现，Sen1的N末端与C末端结构域可介导分子内相互作用。本研究结果为解析调控Sen1介导的非编码转录终止的蛋白质-蛋白质相互作用网络提供了重要理论依据。