Exon repression by polypyrimidine tract binding protein
Exon repression by polypyrimidine tract binding protein
复制标题
DOI：
10.1261/rna.2250405
复制
发表时间：
2005-05-01
期刊：
影响因子：
4.5
通讯作者：
Black, DL
中科院分区：
文献类型：
--
作者：
Amir-Ahmady, B;Boutz, PL;Black, DL
多嘧啶结合蛋白(PTB)可以沉默许多可选外显子的剪接。然而，PTB抑制的外显子还受到其他RNA调节元件和蛋白质的影响。这使得很难剖析沉默剪接的前mRNP复合体的结构，也很难理解PTB在这一过程中的作用。我们确定了PTB介导的剪接抑制的最低要求。我们发现，PTB高亲和力结合的最小序列相对较大，包含多个多嘧啶元件。超速离心法和蛋白水解图分析表明，大多数PTB蛋白以单体形式存在，并且一个多嘧啶基元跨越多个PTB结构域。高亲和力部位最初由PTB单体结合，在较高浓度时由额外的PTB分子结合。值得注意的是，当放置在强测试外显子的3‘剪接位点时，该位点不足以进行剪接抑制。有效的抑制需要在外显子本身或其下游有第二个结合位点。这第二个位点促进了多聚体PTB复合体的形成，即使它本身不能很好地与PTB结合。这些实验表明，PTB足以抑制一个原本构成外显子的剪接，而不需要额外的调节蛋白结合部位，也不会与U2AF结合竞争。介导PTB剪接抑制的最小复合体需要两个与低聚PTB复合体结合的结合位点。
Polypyrimidine tract binding protein (PTB) is known to silence the splicing of many alternative exons. However, exons repressed by PTB are affected by other RNA regulatory elements and proteins. This makes it difficult to dissect the structure of the pre-mRNP complexes that silence splicing, and to understand the role of PTB in this process. We determined the minimal requirements for PTB-mediated splicing repression. We find that the minimal sequence for high affinity binding by PTB is relatively large, containing multiple polypyrimidine elements. Analytical ultracentrifugation and proteolysis mapping of RNA cross-links on the PTB protein indicate that most PTB exists as a monomer, and that a polypyrimidine element extends across multiple PTB domains. The high affinity site is bound initially by a PTB monomer and at higher concentrations by additional PTB molecules. Significantly, this site is not sufficient for splicing repression when placed in the 3' splice site of a strong test exon. Efficient repression requires a second binding site within the exon itself or downstream from it. This second site enhances formation of a multimeric PTB complex, even if it does not bind well to PTB on its own. These experiments show that PTB can be sufficient to repress splicing of an otherwise constitutive exon, without binding sites for additional regulatory proteins and without competing with U2AF binding. The minimal complex mediating splicing repression by PTB requires two binding sites bound by an oligomeric PTB complex.