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Abstract TNFRSF10A (tumor necrosis factor receptor superfamily member 10A) encodes a cell surface receptor protein involved in apoptotic, necroptotic, and inflammatory pathways. Dysregulation of TNFRSF10A has been implicated in sensitization to apoptosis and to the development of multiple diseases, yet little is known of the AC100861.1 long noncoding RNA (lncRNA) that lies head-to-head with TNFRSF10A. Given its genomic positioning, we sought to investigate the function of AC100861.1, focusing on its potential relationship with TNFRSF10A and the role it may play in death receptor signaling. Using knockdown and overexpression strategies, we probed cell viability and examined transcript and protein-level changes in key genes involved in apoptosis, necroptosis, and inflammation. Decreased cell viability was observed upon TNFRSF10A overexpression, regardless of whether the cells were subjected to the chemical stressor tunicamycin. Similarly, overexpression of AC100861.1 led to increased cell death, with a further increase observed under conditions of cellular stress. Knockdown of TNFRSF10A increased cell death only when the cells were stressed, and AC100861.1 knockdown exhibited no effect on cell death. Neither knockdown nor overexpression of either of these genes greatly affected the expression of the other. Manipulating AC100861.1, however, led to marked changes in the expression of genes involved in necroptosis and inflammatory cell-signaling pathways. Additionally, RNA fluorescence in situ hybridization (RNA-FISH) revealed that the AC100861.1 transcript is localized primarily to the cytoplasm. Together, these data suggest that AC100861.1 may have a role in regulating necroptotic and inflammatory signaling pathways and that this function is separate from changes in TNFRSF10A expression. Given the importance of this genomic locus for cell survival, these data provide insight into the function of a poorly understood lncRNA with potential implications regarding disease pathology and treatment.

Abstract Background Long noncoding RNAs (lncRNAs) are emerging as a class of genes whose importance has yet to be fully realized. It is becoming clear that the primary function of lncRNAs is to regulate gene expression, and they do so through a variety of mechanisms that are critically tied to their subcellular localization. Although most lncRNAs are poorly understood, mapping lncRNA subcellular localization can provide a foundation for understanding these mechanisms. Results Here, we present an initial step toward uncovering the localization landscape of lncRNAs in the human retinal pigment epithelium (RPE) using high throughput RNA-Sequencing (RNA-Seq). To do this, we differentiated human induced pluripotent stem cells (iPSCs) into RPE, isolated RNA from nuclear and cytoplasmic fractions, and performed RNA-Seq on both. Furthermore, we investigated lncRNA localization changes that occur in response to oxidative stress. We discovered that, under normal conditions, most lncRNAs are seen in both the nucleus and the cytoplasm to a similar degree, but of the transcripts that are highly enriched in one compartment, far more are nuclear than cytoplasmic. Interestingly, under oxidative stress conditions, we observed an increase in lncRNA localization in both nuclear and cytoplasmic fractions. In addition, we found that nuclear localization was partially attributable to the presence of previously described nuclear retention motifs, while adenosine to inosine (A-to-I) RNA editing appeared to play a very minimal role. Conclusions Our findings map lncRNA localization in the RPE and provide two avenues for future research: 1) how lncRNAs function in the RPE, and 2) how one environmental factor, in isolation, may potentially play a role in retinal disease pathogenesis through altered lncRNA localization.

Formation of new synapses or maintenance of existing synapses requires the delivery of synaptic components from the soma to the nerve termini via axonal transport. One pathway that is important in synapse formation, maintenance and function of the Drosophila neuromuscular junction (NMJ) is the bone morphogenetic protein (BMP)-signaling pathway. Here we show that perturbations in axonal transport directly disrupt BMP signaling, as measured by its downstream signal, phospho Mad (p-Mad). We found that components of the BMP pathway genetically interact with both kinesin-1 and dynein motor proteins. Thick vein (TKV) vesicle motility was also perturbed by reductions in kinesin-1 or dynein motors. Interestingly, dynein mutations severely disrupted p-Mad signaling while kinesin-1 mutants showed a mild reduction in p-Mad signal intensity. Similar to mutants in components of the BMP pathway, both kinesin-1 and dynein motor protein mutants also showed synaptic morphological defects. Strikingly TKV motility and p-Mad signaling were disrupted in larvae expressing two human disease proteins; expansions of glutamine repeats (polyQ77) and human amyloid precursor protein (APP) with a familial Alzheimer's disease (AD) mutation (APPswe). Consistent with axonal transport defects, larvae expressing these disease proteins showed accumulations of synaptic proteins along axons and synaptic abnormalities. Taken together our results suggest that similar to the NGF-TrkA signaling endosome, a BMP signaling endosome that directly interacts with molecular motors likely exist. Thus problems in axonal transport occurs early, perturbs BMP signaling, and likely contributes to the synaptic abnormalities observed in these two diseases.