National Centre for Biomolecular Research, Central European Institute of Technology, Faculty of Science, Masaryk University, Brno CZ-62500, Czechia.
J Biol Chem. 2011 Feb 4;286(5):3645-57. doi: 10.1074/jbc.M110.158774. Epub 2010 Nov 17.
Non-coding RNA polymerase II transcripts are processed by the poly(A)-independent termination pathway that requires the Nrd1 complex. The Nrd1 complex includes two RNA-binding proteins, the nuclear polyadenylated RNA-binding (Nab) 3 and the nuclear pre-mRNA down-regulation (Nrd) 1 that bind their specific termination elements. Here we report the solution structure of the RNA-recognition motif (RRM) of Nab3 in complex with a UCUU oligonucleotide, representing the Nab3 termination element. The structure shows that the first three nucleotides of UCUU are accommodated on the β-sheet surface of Nab3 RRM, but reveals a sequence-specific recognition only for the central cytidine and uridine. The specific contacts we identified are important for binding affinity in vitro as well as for yeast viability. Furthermore, we show that both RNA-binding motifs of Nab3 and Nrd1 alone bind their termination elements with a weak affinity. Interestingly, when Nab3 and Nrd1 form a heterodimer, the affinity to RNA is significantly increased due to the cooperative binding. These findings are in accordance with the model of their function in the poly(A) independent termination, in which binding to the combined and/or repetitive termination elements elicits efficient termination.
非编码 RNA 聚合酶 II 转录本通过不需要多聚（A）的终止途径进行加工，该途径需要 Nrd1 复合物。Nrd1 复合物包括两个 RNA 结合蛋白，核多聚（A）RNA 结合蛋白（Nab）3 和核前体 RNA 下调（Nrd）1，它们结合其特定的终止元件。在这里，我们报告了 Nab3 的 RNA 识别基序（RRM）与 UCUU 寡核苷酸（代表 Nab3 终止元件）复合物的溶液结构。该结构表明，UCUU 的前三个核苷酸位于 Nab3 RRM 的 β-折叠表面上，但仅对中央胞嘧啶和尿嘧啶具有序列特异性识别。我们确定的特异性接触对于体外结合亲和力以及酵母活力都很重要。此外，我们表明，Nab3 和 Nrd1 的两个 RNA 结合基序单独与它们的终止元件的亲和力较弱。有趣的是，当 Nab3 和 Nrd1 形成异二聚体时，由于协同结合，对 RNA 的亲和力显着增加。这些发现与它们在非多聚（A）依赖性终止中的功能模型一致，在该模型中，结合组合和/或重复的终止元件引发有效的终止。
