Nakel Katharina, Bonneau Fabien, Eckmann Christian R, Conti Elena
Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany;
Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany.
Proc Natl Acad Sci U S A. 2015 Jul 14;112(28):8614-9. doi: 10.1073/pnas.1504648112. Epub 2015 Jun 29.
The Caenorhabditis elegans germ-line development defective (GLD)-2-GLD-3 complex up-regulates the expression of genes required for meiotic progression. GLD-2-GLD-3 acts by extending the short poly(A) tail of germ-line-specific mRNAs, switching them from a dormant state into a translationally active state. GLD-2 is a cytoplasmic noncanonical poly(A) polymerase that lacks the RNA-binding domain typical of the canonical nuclear poly(A)-polymerase Pap1. The activity of C. elegans GLD-2 in vivo and in vitro depends on its association with the multi-K homology (KH) domain-containing protein, GLD-3, a homolog of Bicaudal-C. We have identified a minimal polyadenylation complex that includes the conserved nucleotidyl-transferase core of GLD-2 and the N-terminal domain of GLD-3, and determined its structure at 2.3-Å resolution. The structure shows that the N-terminal domain of GLD-3 does not fold into the predicted KH domain but wraps around the catalytic domain of GLD-2. The picture that emerges from the structural and biochemical data are that GLD-3 activates GLD-2 both indirectly by stabilizing the enzyme and directly by contributing positively charged residues near the RNA-binding cleft. The RNA-binding cleft of GLD-2 has distinct structural features compared with the poly(A)-polymerases Pap1 and Trf4. Consistently, GLD-2 has distinct biochemical properties: It displays unusual specificity in vitro for single-stranded RNAs with at least one adenosine at the 3' end. GLD-2 thus appears to have evolved specialized nucleotidyl-transferase properties that match the 3' end features of dormant cytoplasmic mRNAs.
秀丽隐杆线虫生殖系发育缺陷（GLD）-2 - GLD-3复合物上调减数分裂进程所需基因的表达。GLD-2 - GLD-3通过延长生殖系特异性mRNA的短聚腺苷酸尾巴发挥作用，将它们从休眠状态转变为翻译活性状态。GLD-2是一种细胞质非典型聚腺苷酸聚合酶，缺乏典型的细胞核聚腺苷酸聚合酶Pap1所具有的RNA结合结构域。秀丽隐杆线虫GLD-2在体内和体外的活性取决于其与含有多K同源性（KH）结构域的蛋白质GLD-3的结合，GLD-3是双尾C的同源物。我们鉴定出了一个最小的聚腺苷酸化复合物，它包括GLD-2保守的核苷酸转移酶核心和GLD-3的N末端结构域，并以2.3埃的分辨率确定了其结构。该结构表明，GLD-3的N末端结构域并未折叠成预测的KH结构域，而是环绕在GLD-2的催化结构域周围。从结构和生化数据得出的情况是，GLD-3通过稳定酶间接激活GLD-2，并通过在RNA结合裂隙附近提供带正电荷的残基直接激活GLD-2。与聚腺苷酸聚合酶Pap1和Trf4相比，GLD-2的RNA结合裂隙具有不同的结构特征。一致地，GLD-2具有不同的生化特性：它在体外对3'端至少有一个腺苷的单链RNA表现出不同寻常的特异性。因此，GLD-2似乎已经进化出了与休眠细胞质mRNA的3'端特征相匹配的特殊核苷酸转移酶特性。
