ABSTRACT

La-related proteins 7 (LARP7) are a class of RNA chaperones that bind the 3′ ends of RNA and are constitutively associated with their specific target RNAs. In metazoa, Larp7 binds to the long non-coding 7SK RNA as a core component of the 7SK RNP, a major regulator of eukaryotic transcription. In the ciliate Tetrahymena the LARP7 protein p65 is a component of telomerase, an essential ribonucleoprotein complex that maintains the telomeric DNA at eukaryotic chromosome ends. p65 is important for the ordered assembly of telomerase RNA (TER) with telomerase reverse transcriptase. Unexpectedly, Schizosaccharomyces pombe Pof8 was recently identified as a LARP7 protein and a core component of fission yeast telomerase essential for biogenesis. LARP7 proteins have a conserved N-terminal La motif and RRM1 (La module) and C-terminal RRM2 with specific RNA substrate recognition attributed to RRM2, first structurally characterized in p65 as an atypical RRM named xRRM. Here we present the X-ray crystal structure and NMR studies of S. pombe Pof8 RRM2. Sequence and structure comparison of Pof8 RRM2 to p65 and human Larp7 xRRMs reveals conserved features for RNA binding with the main variability in the length of the non-canonical helix α3. This study shows that Pof8 has conserved xRRM features, providing insight into TER recognition and the defining characteristics of the xRRM.

摘要

La 相关蛋白 7 (LARP7) 是一类 RNA 分子伴侣，可结合 RNA 的 3' 端并与其特定的靶标 RNA 组成型相关。在后生动物中，Larp7 与长链非编码 7SK RNA 结合，作为 7SK RNP（真核生物转录的主要调节因子）的核心成分。在纤毛虫四膜虫中，LARP7 蛋白 p65 是端粒酶的一个组成部分，端粒酶是一种必需的核糖核蛋白复合物，在真核染色体末端维持端粒 DNA。p65 对于端粒酶 RNA (TER) 与端粒酶逆转录酶的有序组装很重要。没想到，粟酒裂殖酵母Pof8 最近被确定为 LARP7 蛋白和裂变酵母端粒酶的核心成分，对生物发生至关重要。LARP7 蛋白具有保守的 N 端 La 基序和 RRM1（La 模块）和 C 端 RRM2，具有归因于 RRM2 的特定 RNA 底物识别，首先在 p65 中结构表征为名为 xRRM 的非典型 RRM。在这里，我们展示了S. pombe Pof8 RRM2的 X 射线晶体结构和 NMR 研究。Pof8 RRM2 与 p65 和人类 Larp7 xRRM 的序列和结构比较揭示了 RNA 结合的保守特征，其主要变异是非规范螺旋 α3 的长度。这项研究表明 Pof8 具有保守的 xRRM 特征，提供了对 TER 识别和 xRRM 定义特征的深入了解。