Structural and biochemical analysis of the assembly and function of the yeast pre-mRNA 3′ end processing complex CF I
Structural and biochemical analysis of the assembly and function of the yeast pre-mRNA 3′ end processing complex CF I
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DOI：
10.1073/pnas.1214102110
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发表时间：
2012-12-26
影响因子：
11.1
通讯作者：
Varani, Gabriele
中科院分区：
文献类型：
--
作者：
Barnwal, Ravi Pratap;Lee, Susan D.;Varani, Gabriele
3‘端的切割和多聚腺苷酸化处理的准确性对于mRNA的生物合成和转录终止至关重要。在酵母中，裂解位点上游的两个保守程度较低的相邻元件对于这一过程的准确性和效率非常重要。这两个RNA序列被RNA结合蛋白Hrp1和Rna 15识别，但在体内的有效处理需要一个桥接蛋白(Rna 14)，它与Rna 15形成稳定的异源二聚体二聚体，以稳定RNA-蛋白质复合体。我们早些时候报道了Rna 15和Hrp1与RNA处理元件结合的三元复合体的结构。我们现在报告了在存在和不存在3‘端处理信号的情况下，使用溶液核磁共振研究Hrp1与Rna 14-Rna 15异二聚体的相互作用。通过对Hrp1的甲基选择性标记、体内活性和下拉实验，我们能够研究这个数百kDa的复合体，确定Hrp1内部负责Rna 14募集的界面，并通过结构驱动的突变分析验证这种相互作用的功能意义。
The accuracy of the 3'-end processing by cleavage and polyadenylation is essential for mRNA biogenesis and transcription termination. In yeast, two poorly conserved neighboring elements upstream of cleavage sites are important for accuracy and efficiency of this process. These two RNA sequences are recognized by the RNA binding proteins Hrp1 and Rna15, but efficient processing in vivo requires a bridging protein (Rna14), which forms a stable dimer of heterodimers with Rna15 to stabilize the RNA-protein complex. We earlier reported the structure of the ternary complex of Rna15 and Hrp1 bound to the RNA processing element. We now report the use of solution NMR to study the interaction of Hrp1 with the Rna14-Rna15 heterodimer in the presence and absence of 3'-end processing signals. By using methyl selective labeling on Hrp1, in vivo activity and pull-down assays, we were able to study this complex of several hundred kDa, identify the interface within Hrp1 responsible for recruitment of Rna14 and validate the functional significance of this interaction through structure-driven mutational analysis.