Nair Anil K, Young Matthew A, Menon K M J
Department of Obstetrics/Gynecology, University of Michigan Medical Center, Ann Arbor, MI 48109-0617, USA.
FEBS J. 2008 Jul;275(13):3397-407. doi: 10.1111/j.1742-4658.2008.06490.x. Epub 2008 May 20.
We have shown that hormone-induced downregulation of luteinizing hormone receptor (LHR) in the ovary is post-transcriptionally regulated by an mRNA binding protein. This protein, later identified as mevalonate kinase (MVK), binds to the coding region of LHR mRNA, suppresses its translation, and the resulting ribonucleoprotein complex is targeted for degradation. Mutagenesis and crystallographic studies of rat MVK have established Ser146, Glu193, Asp204 and Lys13 as being crucial for its catalytic function. The present study examined the structural aspects of MVK required for LHR mRNA recognition and translational suppression. Single MVK mutants (S146A, E193Q, D204N and K13A) were overexpressed in 293T cells. Cytosolic fractions were examined for LHR mRNA binding activities by RNA electrophoretic mobility shift analysis. All the single MVK mutants showed decreased LHR mRNA binding activity compared with the wild-type MVK. Double mutants (S146A & E193Q, E193Q & D204N and E193Q & K13A) of MVK also showed a significant decrease in binding to LHR mRNA, suggesting that the residues required for catalytic function are also involved in LHR mRNA recognition. Mutation of the residues outside the catalytic site (D316A and S314A) did not cause any change in LHR mRNA binding activity of MVK when compared with wild-type MVK. To examine the biological effects of these mutants on LHR mRNA expression, a full-length capped rat LHR mRNA was synthesized and translated using a rabbit reticulocyte lysate system in the presence or absence of the MVK mutant proteins. The results showed that mutations of the active site residues of MVK abrogated the inhibitory effect on LHR mRNA translation. Therefore, these data indicate that an intact active site of MVK is required for its binding to rat LHR mRNA and for its translational suppressor function.
我们已经表明，激素诱导的卵巢中促黄体生成素受体（LHR）的下调是由一种mRNA结合蛋白在转录后进行调控的。这种蛋白后来被鉴定为甲羟戊酸激酶（MVK），它与LHR mRNA的编码区结合，抑制其翻译，并且所形成的核糖核蛋白复合物会被靶向降解。对大鼠MVK的诱变和晶体学研究已确定Ser146、Glu193、Asp204和Lys13对其催化功能至关重要。本研究考察了MVK识别LHR mRNA和抑制翻译所需的结构方面。单个MVK突变体（S146A、E193Q、D204N和K13A）在293T细胞中过表达。通过RNA电泳迁移率变动分析检测胞质组分中LHR mRNA的结合活性。与野生型MVK相比，所有单个MVK突变体的LHR mRNA结合活性均降低。MVK的双突变体（S146A&E193Q、E193Q&D204N和E193Q&K13A）与LHR mRNA的结合也显著减少，这表明催化功能所需的残基也参与LHR mRNA的识别。与野生型MVK相比，催化位点外残基的突变（D316A和S314A）未导致MVK的LHR mRNA结合活性发生任何变化。为了考察这些突变体对LHR mRNA表达的生物学效应，在存在或不存在MVK突变蛋白的情况下，使用兔网织红细胞裂解物系统合成并翻译全长加帽的大鼠LHR mRNA。结果表明，MVK活性位点残基的突变消除了对LHR mRNA翻译的抑制作用。因此，这些数据表明MVK完整的活性位点是其与大鼠LHR mRNA结合及其翻译抑制功能所必需的。
