Structure and function of 5' to 3' exoribonucleases

描述(由申请人提供)： 5‘-3’外切核糖核酸酶(XRN)是真核生物中一类保守的酶，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、运输、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，XRN1(175kD)主要是胞质的，参与去去掉的mRNAs的降解，无义介导的衰退，microRNA的衰退，对正常发育是必不可少的。XRN2(115kD)主要是核的，它在酿酒酵母中的同源基因通常被称为Rat1。XRN2/Rat1也是Pol II和Pol I终止转录的鱼雷模型的核心。 在酵母中，RAT1是一个必不可少的基因，而XRN1的缺失会导致生长缓慢和许多其他缺陷。Rat1与另一种蛋白Rai1(45kD)共纯化，在体外可使Rat1的外切核糖核酸酶活性提高约50%。Rai1的弱序列同源基因Dom3Z存在于哺乳动物中，尽管它不与XRN2相互作用。此外，酿酒酵母含有弱的Rai1同源物Ydr370C，但它对Rat1活性没有影响。为了开始了解这些核酸外切酶功能的分子基础，我们测定了S.pombe Rat1-Rai1复合体和K.lactis Xrn1的晶体结构，并根据结构信息进行了生化和功能研究。令我们惊讶的是，晶体结构表明Rai1是一种酶，我们的生化研究证实它具有RNA5‘焦磷水解酶(PPH)活性和解帽活性(但仅针对未甲基化的帽)。我们的进一步研究表明，Dom3Z具有PPH、解帽和分布的5‘-3’外切核糖核酸酶活性，我们将其更名为DXO。因此，我们在哺乳动物中发现了一种新的5‘-3’核酸外切酶。我们还发现Ydr370C具有解壳和5‘-3’外切酶活性，但不具有PPH活性，并将其命名为Dxo1。基于这些生化活性，我们推测DXO家族酶在RNA5‘封端质量控制中起着重要作用。我们在酵母和哺乳动物细胞中的初步功能研究表明，与该领域以前的看法不同，存在不完全的mRNA5‘封顶，以及DXO家族酶在从细胞中清除此类RNA的重要性。这些初步的结构、生化和功能研究为进一步研究经典的XRN和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.