Hiriart Edwige, Gruffat Henri, Buisson Monique, Mikaelian Ivan, Keppler Selina, Meresse Patrick, Mercher Thomas, Bernard Olivier A, Sergeant Alain, Manet Evelyne
INSERM U412, ENS-Lyon, IFR 128 Biosciences Lyon Gerland, Laboratoire de Virologie Humaine, 46 Allée d'Italie, 69364 Lyon, France.
J Biol Chem. 2005 Nov 4;280(44):36935-45. doi: 10.1074/jbc.M501725200. Epub 2005 Aug 29.
The Epstein-Barr virus early protein EB2 (also called BMLF1, Mta, or SM), a protein absolutely required for the production of infectious virions, shares properties with mRNA export factors. By using a yeast two-hybrid screen, we have identified the human protein OTT3 as an EB2-interacting factor. OTT3 is a new member of the Spen (split end) family of proteins (huSHARP, huOTT1, DmSpen, and muMINT), which are characterized by several N-terminal RNA recognition motifs and a highly conserved C-terminal SPOC (Spen Paralog and Ortholog C-terminal) domain that, in the case of SHARP, has been shown to interact with SMRT/NCoR corepressors. OTT3 is ubiquitously expressed as a 120-kDa protein. Transfected OTT3 is a nonshuttling nuclear protein that co-localizes with co-transfected EB2. We also showed that EB2 interacts with the SPOC domains of both OTT1 and SHARP proteins. Although the OTT3 interaction domain maps within the 40 N-terminal amino acids of EB2, OTT1 and SHARP interact within the C-terminal half of the protein. Furthermore, we demonstrated that the capacity of the OTT3 and OTT1 SPOC domains to interact with SMRT and repress transcription is far weaker than that of SHARP. Thus there is no evidence for a role of OTT3 in transcriptional regulation. Most interestingly, however, we have found that OTT3 has a role in splicing regulation; OTT3 represses accumulation of the alternatively spliced beta-thalassemia mRNAs, but it has no effect on the beta-globin constitutively spliced mRNA. Thus our results suggested a new function for Spen proteins related to mRNA export and splicing.
爱泼斯坦-巴尔病毒早期蛋白EB2（也称为BMLF1、Mta或SM）是产生感染性病毒粒子绝对必需的一种蛋白质，它具有与mRNA输出因子相同的特性。通过酵母双杂交筛选，我们鉴定出人类蛋白OTT3是一种与EB2相互作用的因子。OTT3是Spen（分裂末端）蛋白家族（huSHARP、huOTT1、DmSpen和muMINT）的新成员，该家族蛋白的特征是有几个N端RNA识别基序和一个高度保守的C端SPOC（Spen旁系同源物和直系同源物C端）结构域，就SHARP而言，已证明该结构域可与SMRT/NCoR共抑制因子相互作用。OTT3以120 kDa的蛋白质形式普遍表达。转染后的OTT3是一种非穿梭核蛋白，与共转染的EB2共定位。我们还表明，EB2与OTT1和SHARP蛋白的SPOC结构域相互作用。尽管OTT3的相互作用结构域位于EB2的40个N端氨基酸内，但OTT1和SHARP在该蛋白的C端一半内相互作用。此外，我们证明OTT3和OTT1的SPOC结构域与SMRT相互作用并抑制转录的能力远弱于SHARP。因此，没有证据表明OTT3在转录调控中起作用。然而，最有趣的是，我们发现OTT3在剪接调控中起作用；OTT3抑制β地中海贫血可变剪接mRNA的积累，但对β珠蛋白组成性剪接mRNA没有影响。因此，我们的结果提示Spen蛋白与mRNA输出和剪接相关的新功能。
