Department of Biochemistry, Tufts University School of Medicine and the Sackler Graduate School of Biomedical Sciences, Boston, Massachusetts 02111, USA.
RNA. 2011 Apr;17(4):652-64. doi: 10.1261/rna.2273111. Epub 2011 Jan 31.
Synthesis of the poly(A) tail of mRNA in Saccharomyces cerevisiae requires recruitment of the polymerase Pap1 to the 3' end of cleaved pre-mRNA. This is made possible by the tethering of Pap1 to the Cleavage/Polyadenylation Factor (CPF) by Fip1. We have recently reported that Fip1 is an unstructured protein in solution, and proposed that it might maintain this conformation as part of CPF, when bound to Pap1. However, the role that this feature of Fip1 plays in 3' end processing has not been investigated. We show here that Fip1 has a flexible linker in the middle of the protein, and that removal or replacement of the linker affects the efficiency of polyadenylation. However, the point of tethering is not crucial, as a fusion protein of Pap1 and Fip1 is fully functional in cells lacking genes encoding the essential individual proteins, and directly tethering Pap1 to RNA increases the rate of poly(A) addition. We also find that the linker region of Fip1 provides a platform for critical interactions with other parts of the processing machinery. Our results indicate that the Fip1 linker, through its flexibility and protein/protein interactions, allows Pap1 to reach the 3' end of the cleaved RNA and efficiently initiate poly(A) addition.
在酿酒酵母中，mRNA 的 poly(A) 尾巴的合成需要将聚合酶 Pap1 募集到切割前体 mRNA 的 3' 端。这是通过 Fip1 将 Pap1 与切割/多聚腺苷酸化因子 (CPF) 连接起来实现的。我们最近报道称，Fip1 在溶液中是一种无结构的蛋白质，并提出当它与 Pap1 结合时，它可能保持这种构象作为 CPF 的一部分。然而，Fip1 的这一特性在 3' 端加工中所起的作用尚未得到研究。我们在这里表明，Fip1 蛋白的中部有一个灵活的连接子，并且该连接子的缺失或替换会影响多聚腺苷酸化的效率。然而，连接点并不关键，因为缺乏编码必需单个蛋白的基因的细胞中，Pap1 和 Fip1 的融合蛋白是完全有功能的，并且直接将 Pap1 连接到 RNA 上会增加 poly(A) 添加的速率。我们还发现，Fip1 的连接子区域为与加工机制的其他部分进行关键相互作用提供了一个平台。我们的结果表明，Fip1 的连接子通过其灵活性和蛋白质/蛋白质相互作用，允许 Pap1 到达切割 RNA 的 3' 端，并有效地启动 poly(A) 添加。
