Mechanisms of mRNA Anchoring and Translation Regulation on the Endoplasmic Reticulum

RNA定位，一种普遍存在的调节mRNA翻译亚细胞位置的细胞策略， 通过公共的、分阶段的机制运行。首先，顺式编码的本地化序列(邮政编码)是 被RNA结合蛋白识别，并组装成翻译沉默的RNP运输 很复杂。RNP复合体然后通过扩散或通过以下方式定位到适当的亚细胞目的地 主动运输，并抛锚。最后，mrna的翻译被解除抑制，局部蛋白质合成。 随之而来。尽管在识别邮政编码信号方面取得了实质性进展，但反式作用RNA结合 蛋白质、分子马达和运输机制，对分子知之甚少 信使核糖核酸的锚定机制，这对维持蛋白质的局部合成至关重要。在……里面 我们对内质网(ER)上的mRNA定位和锚定机制的研究，我们 发现细胞器蛋白质编码的mRNAs直接锚定在内质网细胞膜上。相比之下， 分泌蛋白编码的mRNAs也定位于内质网，并通过翻译间接锚定 内质网结合的核糖体上。我们假设，直接的RNA锚定机制在空间上作用于 协调功能相关基因的合成。确定直接信使核糖核酸的作用机制 锚定到内质网，我们对内质网结合的多核糖体进行蛋白质组相互作用筛选，并鉴定 候选内质网整合膜RNA锚定蛋白。在第一个目标中，功能验证研究 候选的RNA锚定蛋白将被执行。信使核糖核酸同源性、顺式-内质网锚定基序和RNA 候选相互作用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锚定蛋白的功能将进一步 通过使用siRNA敲除的目标mRNA翻译和定位分析验证，以及在哪里 可用的基因敲除动物模型，以确定ER-mRNA在基因表达中直接锚定的作用。 MRNAs可以直接锚定到内质网的发现表明了核糖体的一种新机制 运输到内质网，在那里膜锚定的mRNAs直接招募核糖体进行从头翻译。在……里面 支持这一模型，我们以前报道过，内质网结合的核糖体在从头翻译启动中起作用 并在翻译终止后保持ER关联。从这些观察延伸出来，我们 假设内质网上的翻译在功能上与胞质翻译分开。一个 该模型的初步预测是，ER转换周期的运行不需要强制交换 核糖体亚基与胞浆池。我们建议在第二个目标中检验这一假设，在那里我们将 确定从头翻译起始的亚细胞位置(S)以及翻译在调节 内质网上的核糖体交换。我们期望拟议的研究将揭示新的范式 健康与疾病中信使核糖核酸翻译的亚细胞组织及其调控。

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.