Mechanisms of mRNA Anchoring and Translation Regulation on the Endoplasmic Reticulum

RNA定位，一种普遍存在的调节mRNA翻译亚细胞位点的细胞策略， 通过一个共同的、分级的机制运行。首先，将顺式编码的定位序列（“邮政编码”） 被RNA结合蛋白识别，mRNA组装成一个功能性沉默的RNP转运体 复杂.然后，RNP复合物通过扩散或通过免疫细胞化学定位于适当的亚细胞目的地。 主动运输，并锚定。最后，mRNA的翻译被解除抑制， 包围尽管在识别邮政编码信号方面已经取得了实质性进展，但反式作用RNA结合 蛋白质，分子马达和运输机制，很少有人知道关于分子 mRNA锚定机制，这对维持局部蛋白质合成至关重要。在 我们对mRNA在内质网（ER）上的定位和锚定机制的研究， 发现细胞器蛋白编码mRNA直接锚定在ER膜上。与此相反， 分泌蛋白编码mRNA，也定位于ER，并通过翻译间接锚定 ER结合的核糖体上。我们假设直接的RNA锚定机制在空间上起作用， 协调功能相关基因的合成。为了确定直接mRNA的作用机制， 锚定到ER，我们进行了ER结合的多聚核糖体的蛋白质组相互作用筛选， 候选ER整合膜RNA锚定蛋白。在第一个目标中， 将进行候选RNA锚定蛋白。mRNA身份、顺式ER锚定基序和RNA 候选相互作用物的结合域将通过光交联和免疫沉淀/RNA- Seq（CLIP-Seq）和PAR-CLIP方法。候选ER-RNA锚定蛋白功能将进一步研究 通过使用siRNA敲低的靶mRNA翻译和定位测定法进行验证， 可用的基因敲除动物模型，以确定直接ER-mRNA锚定在基因表达中的作用。 mRNA可以直接锚定在内质网上的发现提示了核糖体的一种新机制 转运到ER，在ER中膜锚定的mRNA直接募集核糖体用于从头翻译。在 支持这一模型，我们以前报道ER结合的核糖体在从头翻译起始中起作用 并在翻译终止后保持ER相关。根据这些观察，我们 假设ER上的翻译功能上与胞质翻译分开。一 该模型的主要预测是ER翻译循环在没有强制性交换的情况下运行。 核糖体亚基与胞质池。我们建议在第二个目标中测试这个假设，在那里我们将 确定从头翻译起始的亚细胞位点和翻译在调节 核糖体在内质网上的交换我们预计拟议的研究将揭示新的范式 mRNA翻译的亚细胞组织及其在健康和疾病中的调节。

RNA localization, a ubiquitous cellular strategy for regulating the subcellular site of mRNA translation, operates via a common, staged mechanism. First, a cis-encoded localization sequence (“zipcode”) is recognized by RNA-binding proteins and the mRNA assembled into a translationally-silenced RNP transport complex. The RNP complex is then localized to the appropriate subcellular destination, either by diffusion or by active transport, and anchored. Lastly, translation of the mRNA is derepressed and local protein synthesis ensues. Although substantial progress has been made in identifying zipcode signals, trans-acting RNA binding proteins, molecular motors and transport mechanisms, very little is known regarding molecular mechanisms of mRNA anchoring, which is critical to the maintence of localized protein synthesis. In our research into mechanisms of mRNA localization and anchoring on the endoplasmic reticulum (ER), we discovered that organelle protein-encoding mRNAs are directly anchored to the ER membrane. In contrast, secretory protein-encoding mRNAs, which also localize to the ER, and are anchored indirectly, via translation on ER-bound ribosomes. We hypothesize that a direct RNA anchoring mechanism acts to spatially coordinate the synthesis of functionally related genes. To identify the mechanism of direct mRNA anchoring to the ER, we performed proteomic interactor screens of ER-bound polyribosomes and identified candidate ER integral membrane RNA anchoring proteins. In a first aim, functional validation studies of candidate RNA anchoring proteins will be performed. mRNA identities, cis-ER anchoring motifs, and RNA binding domains for candidate interactors will be identified via photocrosslinking and immunoprecipitation/RNA- Seq (CLIP-Seq) and PAR-CLIP approaches. Candidate ER-RNA anchoring protein function will be further validated through assays of target mRNA translation and localization, using siRNA knockdown and where available, knockout animal models, to determine roles for direct ER-mRNA anchoring in gene expression. The finding that mRNAs can be directly anchored to the ER suggests a novel mechanism of ribosome trafficking to the ER, where membrane-anchored mRNAs directly recruit ribosomes for de novo translation. In support of this model, we reported previously that ER-bound ribosomes function in de novo translation initiation and remain ER-associated following translation termination. Extending from these observations, we hypothesize that translation on the ER is functionally compartmentalized from cytosolic translation. A primary prediction of this model is that the ER translation cycle operates without an obligatory exchange of ribosomal subunits with a cytosolic pool. We propose to test this hypothesis in a second aim, where we will determine the subcellular site(s) of de novo translation initiation and the role of translation in the regulation of ribosome exchange on the ER. We expect that the proposed research will reveal new paradigms for the subcellular organization of mRNA translation and its regulation in health and disease.