A polyadenylylation-specific RNA-contact site on the surface of the bifunctional vaccinia virus RNA modifying protein VP39 that is distinct from the mRNA 5' end-binding "cleft".
A polyadenylylation-specific RNA-contact site on the surface of the bifunctional vaccinia virus RNA modifying protein VP39 that is distinct from the mRNA 5' end-binding "cleft".
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双功能痘苗病毒 RNA 修饰蛋白 VP39 表面上的聚腺苷酸化特异性 RNA 接触位点与 mRNA 5 末端结合“裂口”不同。
DOI：
10.1006/jmbi.1998.2417
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Gershon,PD
中科院分区：
文献类型：
--
作者：
Deng,L;Johnson,L;Neveu,JM;Hardin,S;Wang,SM;Lane,WS;Gershon,PD
VP39 是一种双功能 mRNA 修饰蛋白，既充当 mRNA 帽特异性 2'-O-甲基转移酶，又充当 VP55（痘苗病毒 Poly(A) 聚合酶催化亚基）的持续合成因子。尽管甲基转移酶功能所需的蛋白质表面区域已明确界定，但尚不清楚蛋白质聚腺苷酸化功能是否需要直接 RNA 接触，如果需要，接触位点可能位于蛋白质表面的何处。在这里，我们表明 VP55-VP39 异二聚体与带有 U2-N25-U 基序的 50 聚体寡核苷酸形成稳定的复合物，而不是与单独的 VP55 形成稳定复合物的最佳的 U2-N15-U 基序。带有 U2-N25-U 基序（其中下游 U 残基被 4thioU 取代）的寡核苷酸可以有效地光交联至 VP39，但仅限于 VP55-VP39 异二聚体。通过末端标记的 VP39 的部分蛋白水解，寡核苷酸光交联位点定位于残基 Lys90 和 Arg122 之间的 VP39 区域。肽微测序和验证性诱变确定 Arg107 的侧链为光交联位点。用赖氨酸取代该残基完全消除了光交联，这与精氨酸在其他 RNA 结合蛋白中已确定的 RNA 结合作用一致。这项研究为 VP39 表面上的聚腺苷酸化特异性 RNA 接触位点提供了明确的证据，该位点与最近晶体学和生化研究中表征的 RNA 结合甲基转移酶“裂缝”不同。
VP39 is a bifunctional mRNA-modifying protein that acts as both an mRNA cap-specific 2′-O-methyltransferase and a processivity factor for VP55, the vaccinia poly(A) polymerase catalytic subunit. Although regions of the protein surface required for methyltransferase function are well defined, it has been unclear whether the protein polyadenylylation function requires direct RNA contact and, if so, where the contact site(s) might be located on the protein surface. Here, we show that the VP55-VP39 heterodimer forms a stable complex with a 50mer oligonucleotide bearing a U2-N25-U motif, as opposed to the U2-N15-U motif that is optimal for stable complex formation with VP55 alone. An oligonucleotide bearing a U2-N25-U motif in which the downstream U residue is replaced with 4thioU can be efficiently photocrosslinked to VP39, but only in the context of the VP55-VP39 heterodimer. By partial proteolysis of end-labeled VP39, the site of oligonucleotide photocrosslinking was localized to the region of VP39 between residues Lys90 and Arg122. Peptide microsequencing and confirmatory mutagenesis identified the side-chain of Arg107 as the photocrosslinking site. Substitution of this residue with lysine abolished photocrosslinking entirely, consistent with the established RNA binding role of arginine in other RNA-binding proteins. This study provides clear evidence for a polyadenylylation-specific RNA-contact site on the surface of VP39, which is distinct from the RNA-binding methyltransferase “cleft” characterized in recent crystallographic and biochemical studies.