Trafficking and translation of mRNA in osteocyte dendrites

项目摘要 骨细胞通过延长长的树突突起在骨骼内形成相互连接的网络 相互连接，连接到血管系统，并连接到骨骼表面的细胞。适当的编队和维护 其中树突对于骨细胞的存活和周围骨的骨细胞维持至关重要。 在衰老和许多骨骼疾病中，骨细胞树突的丢失先于骨细胞死亡和骨 脆弱。然而，我们并不完全了解骨细胞形成和维持其功能的机制。 树枝状结构。我们将采取一种创新的方法来提高对骨细胞树突生物学的理解 研究已知在神经元等其他细胞的投射中起作用的分子机制。具体来说，我们将 检测骨细胞将信使核糖核酸运输到树突投射和局部翻译蛋白质的能力。自.以来 我们已经展示了超过400个转录本的强大的树突浓缩，我们的理论基础是 骨细胞投射可能与其他细胞类型的投射共享共同的分子调控机制 调节这个亚细胞室的局部转录组、蛋白质组和功能。错误本地化 信使核糖核酸与从神经退行性疾病到癌症的广泛疾病有关，但 在骨细胞中完全没有被研究过。因此，拟议的研究具有重要意义，因为它将推动 了解骨细胞的分子细胞生物学，可能为骨治疗提供新的靶点 治疗骨质疏松症，改善骨骼脆弱。我们将验证这样的假设，即骨细胞将富含树突的 MRNAs位于树突中，并且mRNAs通过其3‘’中的‘邮政编码’序列被运送到树突 非翻译区(3‘非编码区)。在我们的第一个具体目标中，我们将验证富含顶级树枝晶的本地化 在体内使用骨原位杂交的转录本，然后我们将演示骨细胞的局部翻译 翻译图谱和嘌呤霉素掺入法检测富含树突的mRNAs。我们期待着 树突状细胞定位的转录本编码的蛋白质在骨细胞树突状细胞和 因此将在当地翻译。在我们的第二个具体目标中，我们将识别“邮政编码”本地化序列 在树突丰富的转录本和RNA结合蛋白中，通过使用一种 异源报告分析，然后用质谱仪进行亲和纯化。我们预计有一块碎片 在每个树突丰富的转录本的3‘UTR内，将作为RNA结合蛋白的结合位点 将转录本贩卖到树突。这项工作的长期目标是阐明 树突定位的mRNAs对骨细胞树突状细胞的形成和功能的影响，包括特异性RNA的作用 结合蛋白和局部翻译活性的调节。总之，这项工作将描述一种分子 骨细胞生物学的新机制，为骨细胞维持骨的未来研究提供了信息 提供宝贵的新技术和专业技能培训机会。

Project Summary Osteocytes form an interconnected network within bone by extending long dendritic projections that connect to each other, to the vasculature, and to cells on the bone surface. Proper formation and maintenance of these dendrites are critical for osteocyte viability and for osteocytic maintenance of the surrounding bone. During aging and in many bone diseases, loss of osteocyte dendrites precedes osteocyte death and bone fragility. However, we do not fully understand the mechanisms by which osteocytes form and maintain their dendrites. We will take an innovative approach to improve understanding of osteocyte dendrite biology by studying molecular mechanisms known to act in projections of other cells like neurons. Specifically, we will examine the ability of osteocytes to traffic mRNA to their dendritic projections and locally translate proteins. Since we have already demonstrated robust dendrite enrichment of over 400 mRNA transcripts, our rationale is that osteocyte projections may share a common molecular regulatory mechanism with projections of other cell types to regulate the local transcriptome, proteome, and function of this subcellular compartment. Mis-localization of mRNA has been implicated in a wide range of disorders from neurodegenerative disease to cancer, but is completely unexplored in osteocytes. Therefore, the proposed research is significant because it will advance understanding of molecular cell biology in osteocytes, and it may provide new targets for therapies to treat bone disease and improve bone fragility. We will test the hypothesis that osteocytes translate dendrite-enriched mRNAs locally in dendrites, and that mRNAs are trafficked to dendrites via ‘zip code’ sequences in their 3’ untranslated region (3’UTR). In our first specific aim we will validate localization of top dendrite-enriched transcripts in vivo using in situ hybridization in bone, and then we will demonstrate local translation of osteocyte dendrite-enriched mRNAs using translational profiling and puromycin incorporation assays. We expect that dendrite-localized transcripts encode proteins that play essential roles in the function of osteocyte dendrites and will therefore be locally translated. In our second specific aim, we will identify ‘zip code’ localization sequences in dendrite-enriched transcripts and RNA binding proteins that confer dendrite localization in osteocytes using a heterologous reporter assay and then affinity purification with mass spectrometry. We expect that a fragment within the 3’UTR of each dendrite-enriched transcript will serve as the binding site for an RNA binding protein that trafficks that transcript to dendrites. The long-term goal of this work is to elucidate the contribution of dendrite-localized mRNAs to formation and function of osteocyte dendrites, including the role of specific RNA binding proteins and regulation of local translational activity. Together, this work will characterize a molecular mechanism that is novel to osteocyte cell biology, inform future research on osteocyte maintenance of bone, and provide a valuable training opportunity in new technical and professional skills.