After reeiving the EAPSI award, the Blackshaw lab and Mattick lab continued their collaboration in numerous. As a result of the collaboration enabled by this award, we found numerous regions of lncRNAs important for progenitor cell differentation in the brain including Gomafu and Neat1. We conducted 18 protein microarray assays and followed up on many of these candidates by in situ hybridization in the developing mouse brain. Through our in situ screen, we found a small subset of lncRNAs that are selectively expressed in the post-natal and adult brain. Many of these selectively expressed lncRNAs are opposite strand transcripts (OSTs), which are in close genomic proximity to protein coding genes. Interestingly, although the expression of OST transcripts overlaps with expression of the associated protein coding gene, we find many instances of divergent expression. The vast majority of lncRNAs examined have developmentally dynamic expression patterns and are most highly expressed at embryonic time points. Additionally, we observe expression in numerous embryonic brain structures in both proliferating and postmitotic cells. We further examined a subset of our candidate list biochemically using a full human protein microarray to identify protein binding partners. Utilizing numerous RNA folding algorithms, we determined that some lncRNAs examined by in situ hybridization contained regions of highly conserved secondary structure. By isolating these regions of conservation and hybridizing them to a full human protein array containing 17,000 proteins, we identified numerous putative protein-binding partners of these molecules. Gene Ontology analysis showed that these lncRNA regions of conservation interact with a small number of protein functional classes giving greater insight into the possible role of these molecules in the cell. Taken together, our findings suggest that a great number of lncRNAs are expressed in a developmentally dynamic manner which allows for tight regulation of biochemical interactions and possibly transcriptional regulation throughout neuronal development.
