Müller W E, Slor H, Pfeifer K, Hühn P, Bek A, Orsulic S, Ushijima H, Schröder H C
Institut für Physiologische Chemie, Universität, Mainz, Germany.
J Mol Biol. 1992 Aug 5;226(3):721-33. doi: 10.1016/0022-2836(92)90628-w.
Resealed nuclear envelope (NE) vesicles from rat liver containing entrapped exogenous RNA were used to study the effect of adenosine+uridine binding factor (AUBF), present in cytosolic cell extracts, on ATP-dependent transport of A+U-rich RNA (AU+RNA) and A+U-free RNA (AU-RNA) across the NE. This factor specifically binds to A+U-rich sequences present in the 3' untranslated regions of lymphokine and cytokine mRNAs, containing overlapping AUUUA boxes (granulocyte-macrophage colony stimulating factor, interleukin-3). Addition of AUBF to the extravesicular compartment markedly increased the efflux of the in vitro transcribed, capped and polyadenylated AU+ RNAs. Export of entrapped AU- control RNA, such as beta-globin RNA, was not affected by AUBF, in contrast to chimeric AU+ beta-globin RNA containing the A+U-rich sequence of human interferon-alpha mRNA (6 reiterated AUUUA motifs). Competition experiments revealed that AUBF enhances the affinity of poly(A)-containing AU+ RNAs to the NE poly(A)-binding component (poly(A)-recognizing mRNA carrier p106), and thereby accelerates nuclear export of these RNAs. We could demonstrate that AUBF added to the extravesicular space forms stable complexes with polyadenylated AU+ RNA with relative molecular masses of about 45,000, 62,000 and 70,000 inside the vesicles or during ATP-dependent export. In addition we determined that AUBF may affect mRNA stability by protecting A+U-rich RNA against degradation by trans-acting, nuclear matrix-associated and A+U-specific endoribonuclease V.
来自大鼠肝脏的重新封闭的核膜（NE）囊泡，其中包裹着外源RNA，用于研究存在于细胞质细胞提取物中的腺苷+尿苷结合因子（AUBF）对富含A+U的RNA（AU+RNA）和不含A+U的RNA（AU-RNA）跨核膜的ATP依赖性转运的影响。该因子特异性结合存在于淋巴因子和细胞因子mRNA 3'非翻译区的富含A+U的序列，这些序列包含重叠的AUUUA框（粒细胞-巨噬细胞集落刺激因子、白细胞介素-3）。向囊泡外腔添加AUBF显著增加了体外转录、加帽和聚腺苷酸化的AU+RNA的外排。与含有人类干扰素-α mRNA富含A+U序列（6个重复的AUUUA基序）的嵌合AU+β-珠蛋白RNA相反，包裹的AU-对照RNA（如β-珠蛋白RNA）的输出不受AUBF影响。竞争实验表明，AUBF增强了含poly(A)的AU+RNA与核膜poly(A)结合成分（识别poly(A)的mRNA载体p106）的亲和力，从而加速了这些RNA的核输出。我们可以证明，添加到囊泡外空间的AUBF在囊泡内或ATP依赖性输出过程中与相对分子质量约为45,000、62,000和70,000的聚腺苷酸化AU+RNA形成稳定复合物。此外，我们确定AUBF可能通过保护富含A+U的RNA免受反式作用、与核基质相关的A+U特异性核糖核酸内切酶V的降解来影响mRNA稳定性。
