Structure and function of 5' to 3' exoribonucleases

描述（由申请人提供）： 5 '-3'核糖核酸外切酶（XRN）是真核生物中的一个保守酶家族，在RNA代谢和RNA干扰中具有重要功能，包括核糖体和小核仁RNA的质量控制、降解、转运、成熟以及RNA聚合酶I和II（Pol I和Pol II）的转录终止。在真菌和动物中，XRN 1（175 kD）主要存在于胞质中，参与脱帽mRNA的降解、无义介导的衰变、microRNA衰变，并且对正常发育至关重要。XRN 2（115 kD）主要是核蛋白，在S.酿酒酵母通常被称为Rat 1。XRN 2/Rat 1也是Pol II和Pol I终止转录的鱼雷模型的核心。在 RAT 1是酵母的必需基因，XRN 1的缺失导致生长缓慢和许多其他缺陷。Rat 1与另一种蛋白质Rai 1（45 kD）共纯化，其在体外将Rat 1的核糖核酸外切酶活性提高约50%。Rai 1的一个弱序列同源物，称为Dom 3 Z，存在于哺乳动物中，尽管它不与XRN 2相互作用。此外，S.酿酒酵母含有一个弱的Rai 1同源物，Ydr 370 C，但它对Rat 1活性没有影响。 为了开始理解这些核酸外切酶功能的分子基础，我们已经确定了S. pombe Rat 1-Rai 1复合体和K. lactis Xrn 1的结构信息，并进行了生化和功能研究。令我们惊讶的是，晶体结构表明Rai 1是一种酶，我们的生物化学研究证实它具有RNA 5'焦磷酸水解酶（PPH）活性和去帽活性（但仅针对未甲基化的帽）。我们进一步的研究表明Dom 3 Z具有PPH、脱帽和分布性5 '-3'核糖核酸外切酶活性，我们将其重新命名为DXO。因此，我们在哺乳动物中鉴定了一种新的5 '-3'核酸外切酶。我们还发现Ydr 370 C具有去帽和5 '-3'核酸外切酶活性，但没有PPH活性，并将其命名为Dxo 1。基于这些生物化学活性，我们假设DXO家族酶在RNA 5'加帽质量控制中具有重要作用。我们在酵母和哺乳动物细胞中的初步功能研究已经证明了不完全mRNA 5'加帽的存在，与该领域以前的信念相反，以及DXO家族酶在从细胞中清除此类RNA中的重要性。这些初步的结构、生物化学和功能研究为进一步表征经典XRN以及DXO家族酶奠定了基础。

DESCRIPTION (provided by applicant): Project Summary The 5'-3' exoribonucleases (XRNs) comprise a family of conserved enzymes in eukaryotes with important functions in RNA metabolism and RNA interference, including quality control, degradation, transport, maturation of ribosomal and small nucleolar RNAs, and transcription termination of RNA polymerases I and II (Pol I and Pol II). In fungi and animals, XRN1 (175 kD) is primarily cytosolic and is involved in degradation of decapped mRNAs, nonsense mediated decay, microRNA decay and is essential for proper development. XRN2 (115 kD) is primarily nuclear, and its ortholog in S. cerevisiae is more commonly known as Rat1. XRN2/Rat1 is also central to the torpedo model of transcription termination by Pol II and Pol I. In yeast, RAT1 is an essential gene, and deletion of XRN1 leads to slow growth and many other defects. Rat1 co-purifies with another protein, Rai1 (45 kD), which enhances the exoribonuclease activity of Rat1 by ~50% in vitro. A weak sequence homolog of Rai1, known as Dom3Z, exists in mammals, although it does not interact with XRN2. In addition, S. cerevisiae contains a weak Rai1 homolog, Ydr370C, but it has no effect on Rat1 activity. To begin to understand the molecular basis for the functions of these exonucleases, we have determined the crystal structures of the S. pombe Rat1-Rai1 complex and K. lactis Xrn1, and carried out biochemical and functional studies based on the structural information. To our surprise, the crystal structure suggested that Rai1 is an enzyme, and our biochemical studies confirmed that it has RNA 5' pyrophosphohydrolase (PPH) activity and decapping activity (but only toward unmethylated caps). Our further studies showed that Dom3Z has PPH, decapping, and distributive 5'-3' exoribonuclease activity, and we have renamed it DXO. Therefore, we have identified a new 5'-3' exonuclease in mammals. We also found that Ydr370C has decapping and 5'-3' exonuclease activities but no PPH activity, and have named it Dxo1. We hypothesized based on these biochemical activities that the DXO family enzymes have important roles in RNA 5' capping quality control. Our preliminary functional studies in yeast and mammalian cells have demonstrated the existence of incomplete mRNA 5' capping, in contrast to previous beliefs in the field, and the importance of the DXO family enzymes in clearing such RNAs from the cell. These preliminary structural, biochemical and functional studies have set the stage for further characterizations of the classical XRNs as well as the DXO family enzymes.