Trafficking and translation of mRNA in osteocyte dendrites

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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.