Alvarez Periel Elena
| Alvarez Periel Elena |
Alvarez Periel Elena1, Marianne C. Kramer2, Daniel, P. Jackson1, Cagla Akay-Espinoza1, Julia Tasca3, Benjamin A. Garcia3, Jordan-Sciutto, L. Kelly1, Brian, D Gregory2
1Basic & Translational Sciences, University of Pennsylvania, School of Dental Medicine; 2Department of Biology, Cell and Molecular Biology Graduate Group, University of Pennsylvania; 3Epigenetics Institute, Department of Biochemistry and Biophysics, University of Pennsylvania
Introduction
RNA-binding proteins (RBPs) participate in virtually all steps of RNA metabolism regulation, which is particularly relevant in neurons and in cellular responses to stressors. One important stressor for neurons is excitotoxicity, which is caused by excessive excitatory amino acids signaling leading to intracellular calcium overload and eventually to neuronal death. Importantly, excitotoxicity has been implicated in neuronal dysfunction and degeneration in many pathologies including Alzheimer’s disease (AD), Huntington’s disease (HD) or HIV-associated neurocognitive disorders (HAND), amongst others. However, to our knowledge, global analysis of changes in RNA structure and in protein binding in an excitotoxic context has not been assessed.
Methods
To this end, we aimed to identify global changes in RNA-protein interactions and in RNA secondary structure in an excitotoxicity in vitro model using rat primary cortical neurons. We have applied the novel protein interaction profile sequencing (PIP-seq) method to identify sites within an RNA molecule that are bound by an RNA-binding protein (RBP), termed protein protected sites (PPS).
Results
Our data shows relevant shifts in RNA secondary structure between untreated- and NMDA-treated neurons near the start and stop codons of protein coding genes. Additionally, we have identified sets of genes that have PPSs specifically in the control condition, specifically after NMDA treatment or in both conditions but at different intragenic regions. Interestingly, GO analysis of these gene sets shows that transcripts that are protein-bound only in NMDA-treated samples are associated with several neurodegenerative disorders. Finally, using these data we have found RNA motifs enriched in NMDA-specific PPSs and have used them to identify NMDA-dependent RBPs, which might shed light on RBPs post-transcriptional role in excitotoxic stress.
Conclusion
In sum, we have globally examined RNA secondary structure and the RBP-ome of primary cortical neurons in an in vitro excitotoxic model, and identified specific RBPs participating in this regulation.