TRAPped in Translation: Optimized Astrocytic Ribosome Capture via TRAP Methodology for Translatome Sequencing

Abstract

The central nervous system (CNS) is complex and diverse in structure and function, making it difficult to pinpoint the causes of neurological diseases and disorders. Many genetic disorders arise from poorly understood genetic mutations. Initially, neuroscientists focused on neurons for mutation mining, but evidence now supports glial cells, specifically astrocytes, as a potential candidate. Further research of astrocytic gene expression could uncover the mechanisms underlying these mutations. Previous protocols have lacked specificity when it comes to astrocytic capturing and profiling. To address this problem, we have optimized a previously published protocol, titled translating ribosome affinity purification (TRAP) to isolate and validate the translatomes of astrocytes. Using TRAP, messenger RNA fragments attached to the tagged ribosome is isolated, which corresponds to expression levels of various genes actively being translated. In the optimization, the RPL10A transgene that encodes for a 60S ribosomal subunit protein in astrocytes is tagged with EGFP. The fluorescent tag allows for the capture using antibody coated magnetic beads, followed by purification, quantification, and validation of captured mRNA. Using the optimized protocol for astrocytic ribosomes, quantitative results collected via qPCR runs using specific primers of various genes of interest indicated a five-fold enrichment of ALDH1I1, an astrocytic gene, in the immunoprecipitated fraction versus the unbound. Enrichment of astrocytic genes implies sufficient isolation and validation of the translatomes, thus allowing for bulk transcriptome sequencing and translational profiling. Comparative analysis of the mRNA associated with local translation in Fragile X and Alzheimer's disease models can identify genetic indicators before symptoms develop. Pipeline scheme from brain removal to qPCR analysis of cell type primers. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.