p180 Promotes the Ribosome-Independent Localization of a Subset of mRNA to the Endoplasmic Reticulum

In metazoans, the majority of mRNAs coding for secreted and membrane-bound proteins are translated on the surface of the endoplasmic reticulum (ER). Although the targeting of these transcripts to the surface of the ER can be mediated by the translation of a signal sequence and their maintenance is mediated by interactions between the ribosome and the translocon, it is becoming increasingly clear that additional ER-localization pathways exist. Here we demonstrate that many of these mRNAs can be targeted to, and remain associated with, the ER independently of ribosomes and translation. Using a mass spectrometry analysis of proteins that associate with ER-bound polysomes, we identified putative mRNA receptors that may mediate this alternative mechanism, including p180, an abundant, positively charged membrane-bound protein. We demonstrate that p180 over-expression can enhance the association of generic mRNAs with the ER. We then show that p180 contains a lysine-rich region that can directly interact with RNA in vitro. Finally, we demonstrate that p180 is required for the efficient ER-anchoring of bulk poly(A) and of certain transcripts, such as placental alkaline phosphatase and calreticulin, to the ER. In summary, we provide, to our knowledge, the first mechanistic details for an alternative pathway to target and maintain mRNA at the ER. It is likely that this alternative pathway not only enhances the fidelity of protein sorting, but also localizes mRNAs to various subdomains of the ER and thus contributes to cellular organization.

在多细胞动物体内，绝大多数编码分泌型与膜结合型蛋白质的mRNA均在内质网（endoplasmic reticulum, ER）表面完成翻译。尽管这类转录本向ER表面的靶向过程可通过信号肽的翻译实现，其稳定锚定则依赖于核糖体与易位子之间的相互作用，但越来越多的研究表明，还存在其他的ER定位通路。本研究证实，多数此类mRNA可在不依赖核糖体与翻译过程的情况下，靶向并结合于ER表面。我们通过对ER结合多聚核糖体相关蛋白进行质谱分析，鉴定出了可能介导该替代通路的推定mRNA受体，其中包括p180——一种丰富的带正电荷膜结合蛋白。我们证实，p180过表达可增强普通mRNA与ER的结合能力。随后我们发现，p180含有一个富含赖氨酸的结构域，该结构域可在体外直接与RNA相互作用。最后我们证实，p180对于整体多聚(A) RNA以及胎盘碱性磷酸酶、钙网蛋白等特定转录本的高效ER锚定过程至关重要。综上，据我们所知，本研究首次阐明了一条靶向并锚定mRNA于ER表面的替代通路的机制细节。该替代通路不仅可能提升蛋白质分选的保真度，还可将mRNA定位至ER的不同亚结构域，进而参与细胞组织结构的构建。