Structure and function of 5' to 3' exoribonucleases

描述(申请人提供)：5‘-gt；3’外切核糖核酸酶(XRN)是真核生物中一大类保守的酶，在RNA代谢和RNA干扰中具有重要功能。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，在RNA从细胞核到细胞质的运输以及RNA聚合酶III对某些基因的转录起作用。最近的研究发现XRN2/Rat1是外核糖核酸酶，对于RNA聚合酶II(PolII)终止转录的“鱼雷”模式是必不可少的。在酿酒酵母中，Rat1与另一种蛋白Rai1(Rat1相互作用蛋白1)共同纯化，这似乎在体外增强了Rat1的外切核糖核酸酶活性。Rai1的弱序列同源基因DOM3Z存在于哺乳动物中，尽管它可能与XRN2无关。XRN在RNA代谢、RNA干扰和Pol II终止等方面的重要作用引起了人们的极大兴趣。然而，这些酶的功能的分子机制目前仍然知之甚少。作为填补这些知识空白的第一步，我们最近确定了Rat1-Rai1复合体以及Rai1和DOM3Z的晶体结构，并根据结构信息进行了初步的生化和功能研究。这些结果为进一步研究这些蛋白质的结构、生化和生物学奠定了基础。最有趣的是，我们的初步研究表明，Rai1/DOM3Z含有一个单独的活性部位，具有RNA 5‘焦磷酸水解酶活性，我们推测Rai1/DOM3Z可能在RNA代谢或质量控制中具有重要功能。我们还将在我们提出的研究项目中检验这一假说，这将有助于确定这种新的真核酶的生理功能。

DESCRIPTION (provided by applicant): The 5'->3' exoribonucleases (XRNs) comprise a large family of conserved enzymes in eukaryotes with important functions in RNA metabolism and RNA interference. 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, more commonly known as Rat1, has roles in RNA trafficking from the nucleus to the cytoplasm and transcription of some genes by RNA polymerase III. More recent studies have identified XRN2/Rat1 as the exoribonuclease that is essential for the 'torpedo' model of transcription termination by RNA polymerase II (Pol II). In S. cerevisiae, Rat1 co-purifies with another protein, Rai1 (Rat1 interacting protein 1), which appears to enhance the exoribonuclease activity of Rat1 in vitro. A weak sequence homolog of Rai1, known as DOM3Z, exists in mammals, although it probably does not associate with XRN2. The important functions of the XRNs in RNA metabolism, RNA interference and Pol II termination have generated significant interest in them. However, the molecular mechanism for the functions of these enzymes is currently still poorly understood. As a first step to fill these gaps in our knowledge, we recently determined the crystal structures of the Rat1-Rai1 complex as well as of Rai1 and DOM3Z alone, and carried out preliminary biochemical and functional studies based on the structural information. These results set the stage for further structural, biochemical and biological studies on these proteins. Most interestingly, our initial studies show that Rai1/DOM3Z contains a separate active site, with RNA 5' pyrophosphohydrolase activity, and we hypothesize that Rai1/DOM3Z may have important functions in RNA metabolism or quality control. We will also test this hypothesis in our proposed research project, which will help to define the physiological functions of this novel eukaryotic enzyme.