The crystal structure of S. cerevisiae Sad1, a catalytically inactive deubiquitinase that is broadly required for pre-mRNA splicing.
The crystal structure of S. cerevisiae Sad1, a catalytically inactive deubiquitinase that is broadly required for pre-mRNA splicing.
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DOI：
10.1261/rna.042838.113
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发表时间：
2014-05
期刊：
影响因子：
--
通讯作者：
Guthrie C
中科院分区：
文献类型：
--
作者：
Hadjivassiliou H;Rosenberg OS;Guthrie C
泛素相关结构域存在于许多剪接体蛋白中，但它们所起的作用在很大程度上还不清楚。这项研究对SAD1的功能提供了有趣的见解，它是一种剪接体蛋白，包含一个锌指泛素结合域和一个非催化泛素特异的蛋白酶结构域。这项工作表明，SAD1是体内前mRNA剪接和剪接体组装所必需的。出乎意料的是，SAD1在体外无法结合泛素。通过确定SAD1的晶体结构，作者证明了SAD1结构域如何确实偏离了典型的泛素结合域。SAD1是一种重要的剪接因子，最初在酿酒酵母的遗传筛查中被确定为SnRNP组装缺陷的基因。根据序列同源性，SAD1或人类的USP39被预测包括两个结构域：锌指泛素结合域(ZnF-UBP)和非活性泛素特异蛋白酶(IUSP)结构域，这两个结构域都很保守。这些结构域在剪接中的作用以及它们与泛素的相互作用尚不清楚。我们首先使用剪接微阵列分析了SAD1在体内的功能，发现SAD1对几乎所有酵母内含子基因的剪接都是至关重要的。通过体外实验，我们证明了它是组装活性剪接体所必需的。为了获得SAD1功能的结构洞察力，我们确定了全长蛋白质的晶体结构，分辨率为1.8？在结构中，iUSP结构域形成了特征的泛素结合口袋，尽管活性部位的氨基酸取代导致结构域的酶活性完全失活。SAD1锌F-UBP结构域与其他锌F-UBP结构相似，但S锌氟-UBP不具有典型的泛素结合基序。考虑到锌F-UBP结构域作为邻近USP结构域的激活剂的先例，我们认为SAD‘S锌F-UBP以泛素不依赖的能力招募和/或激活SAD’S的iUSP结构域与剪接体相互作用。
Ubiquitin-related domains are present in many spliceosomal proteins, but the roles they play remain largely unknown. This study provides intriguing insights into the function of Sad1, a spliceosomal protein that contains both a Zn finger ubiquitin binding domain and a noncatalytic ubiquitin-specific protease domain. This work demonstrates that Sad1 is required for pre-mRNA splicing in vivo and for spliceosome assembly. Unexpectedly, Sad1 is unable to bind ubiquitin in vitro. By determining the crystal structure of Sad1, the authors show how the Sad1 domains indeed deviate from canonical ubiquitin binding domains. Sad1 is an essential splicing factor initially identified in a genetic screen in Saccharomyces cerevisiae for snRNP assembly defects. Based on sequence homology, Sad1, or USP39 in humans, is predicted to comprise two domains: a zinc finger ubiquitin binding domain (ZnF-UBP) and an inactive ubiquitin-specific protease (iUSP) domain, both of which are well conserved. The role of these domains in splicing and their interaction with ubiquitin are unknown. We first used splicing microarrays to analyze Sad1 function in vivo and found that Sad1 is critical for the splicing of nearly all yeast intron-containing genes. By using in vitro assays, we then showed that it is required for the assembly of the active spliceosome. To gain structural insights into Sad1 function, we determined the crystal structure of the full-length protein at 1.8 Å resolution. In the structure, the iUSP domain forms the characteristic ubiquitin binding pocket, though with an amino acid substitution in the active site that results in complete inactivation of the enzymatic activity of the domain. The ZnF-UBP domain of Sad1 shares high structural similarly to other ZnF-UBPs; however, Sad1's ZnF-UBP does not possess the canonical ubiquitin binding motif. Given the precedents for ZnF-UBP domains to function as activators for their neighboring USP domains, we propose that Sad1's ZnF-UBP acts in a ubiquitin-independent capacity to recruit and/or activate Sad1's iUSP domain to interact with the spliceosome.
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影响因子：
64.5
作者：
Köhler A;Zimmerman E;Schneider M;Hurt E;Zheng N
通讯作者：
Zheng N
影响因子：
5.3
作者：
Lee, KK;Florens, L;Workman, JL
通讯作者：
Workman, JL
影响因子：
64.8
作者：
Mishra, Shravan Kumar;Ammon, Tim;Popowicz, Grzegorz M.;Krajewski, Marcin;Nagel, Roland J.;Ares, Manuel, Jr.;Holak, Tad A.;Jentsch, Stefan
通讯作者：
Jentsch, Stefan
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH
影响因子：
16.8
作者：
Bellare, Priya;Small, Eliza C.;Sontheimer, Erik J.
通讯作者：
Sontheimer, Erik J.