Yi Rui, Bogerd Hal P, Wiegand Heather L, Cullen Bryan R
Department of Genetics, Duke University Medical Center, Durham, North Carolina 27710, USA.
RNA. 2002 Feb;8(2):180-7. doi: 10.1017/s1355838202012384.
The Ran protein regulates nucleocytoplasmic transport mediated by the karyopherin family of nuclear transport factors. Ran is converted to the active, GTP bound form in the nucleus and then binds to a conserved domain found in all karyopherins. This interaction induces cargo binding for exportins and cargo release for importins. In either case, the Ran.GTP is then transported to the cytoplasm by the karyopherin, where it is hydrolyzed to Ran.GDP. To ask whether Ran could function as a nuclear mRNA export factor, we fused Ran to the MS2 coat protein and inserted MS2 RNA-binding sites into an unspliced cat mRNA that is normally sequestered in the nucleus. Coexpression of MS2-Ran induced cat mRNA export and CAT enzyme expression as effectively as, for example, an MS2-Rev fusion protein. MS2-Ran dependent nuclear mRNA export was reduced by inhibitors specific for Crm1, but not blocked as was seen with MS2-Rev. Consistent with the hypothesis that Crm1 is not the only karyopherin cofactor for MS2-Ran mediated mRNA export, we show that not only Crm1 but also CAS, transportin, importin beta and exportin t can all export mRNA from the nucleus when tethered via the MS2 RNA-binding domain. In contrast, two shuttling hnRNPs, hnRNP A1 and hnRNP K, proved unable to function as nuclear RNA export factors when expressed as MS2 fusions. Together, these data argue that karyopherins that normally function to transport proteins into or out of the nucleus are also capable of exporting tethered mRNA molecules.
Ran蛋白调节由核转运因子的核转运蛋白家族介导的核质运输。Ran在细胞核中转化为活性的、结合GTP的形式,然后与所有核转运蛋白中发现的保守结构域结合。这种相互作用诱导货物与输出蛋白结合以及货物从输入蛋白释放。在任何一种情况下,Ran·GTP随后都由核转运蛋白转运到细胞质中,在那里它被水解为Ran·GDP。为了探究Ran是否可以作为一种核mRNA输出因子,我们将Ran与MS2外壳蛋白融合,并将MS2 RNA结合位点插入到通常被隔离在细胞核中的未剪接的猫白血病病毒(cat)mRNA中。MS2-Ran的共表达诱导cat mRNA输出和CAT酶表达的效果与例如MS2-Rev融合蛋白一样有效。特异性针对Crm1的抑制剂降低了MS2-Ran依赖的核mRNA输出,但不像MS2-Rev那样被阻断。与Crm1不是MS2-Ran介导的mRNA输出的唯一核转运蛋白辅因子这一假设一致,我们表明,当通过MS2 RNA结合结构域连接时,不仅Crm1而且CAS、运输蛋白、输入蛋白β和输出蛋白t都可以将mRNA从细胞核中输出。相比之下,当作为MS2融合蛋白表达时,两种穿梭hnRNP,hnRNP A1和hnRNP K被证明不能作为核RNA输出因子发挥作用。总之,这些数据表明,通常负责将蛋白质转运进或转运出细胞核的核转运蛋白也能够输出连接的mRNA分子。