Triqueneaux G, Velten M, Franzon P, Dautry F, Jacquemin-Sablon H
CNRS UPR 9044, Génétique Moléculaire et Intégration des Fonctions Cellulaires, Institut de Recherches sur le Cancer, BP 8, 94801 Villejuif, France.
Nucleic Acids Res. 1999 Apr 15;27(8):1926-34. doi: 10.1093/nar/27.8.1926.
The human unr gene encodes an 85 kDa protein which contains five cold shock domains (CSD). The capacity of Unr to interact in vitro with RNA and its intracellular localization suggest that Unr could be involved in some aspect of cytoplasmic mRNA metabolism. As a step towards identification of Unr mRNA targets, we investigated the RNA-binding specificity of Unr by an in vitro selection approach (SELEX). Purine-rich sequences were selected by Unr, leading to the identification of two related consensus sequences characterized by a conserved core motif AAGUA/G or AACG downstream of a purine stretch. These consensus sequences are 11-14 nt long and appear unstructured. RNAs containing a consensus sequence were bound specifically by Unr with an apparent dissociation constant of 1 x 10(-8) M and both elements, the 5' purine stretch and the core motif, were shown to contribute to the high affinity. When the N-terminal and C-terminal CSD were analyzed individually, they exhibited a lower affinity than Unr for winner sequences (5- and 100-fold, respectively) but with similar binding specificity. Two combinations of CSDs, CSD1-2-3 and CSD1*2-3-4-5 were sufficient to achieve the high affinity of Unr, indicating some redundancy between the CSDs of Unr for RNA recognition. The SELEX-generated consensus motifs for Unr differ from the AACAUC motif selected by the Xenopus Y-box factor FRGY2, indicating that a diversity of RNA sequences could be recognized by CSD-containing proteins.
人类unr基因编码一种85 kDa的蛋白质，该蛋白质包含五个冷休克结构域（CSD）。Unr在体外与RNA相互作用的能力及其细胞内定位表明，Unr可能参与细胞质mRNA代谢的某些方面。作为鉴定Unr mRNA靶标的第一步，我们通过体外筛选方法（SELEX）研究了Unr的RNA结合特异性。Unr选择了富含嘌呤的序列，从而鉴定出两个相关的共有序列，其特征是在嘌呤延伸下游有一个保守的核心基序AAGUA/G或AACG。这些共有序列长度为11 - 14 nt，且呈非结构化。含有共有序列的RNA与Unr特异性结合，表观解离常数为1×10^(-8) M，并且5'嘌呤延伸和核心基序这两个元件均显示对高亲和力有贡献。当分别分析N端和C端的CSD时，它们对获胜序列的亲和力低于Unr（分别低5倍和100倍），但具有相似的结合特异性。CSD的两种组合，即CSD1 - 2 - 3和CSD1*2 - 3 - 4 - 5足以实现Unr的高亲和力，这表明Unr的CSD在RNA识别方面存在一些冗余。通过SELEX生成的Unr共有基序与非洲爪蟾Y盒因子FRGY2选择的AACAUC基序不同，这表明含CSD的蛋白质可以识别多种RNA序列。
