Our efforts to delineate the biochemical processes that convert a transient experience to a persistent memory led to the discovery that functional aggregation of a Drosophila RNA-binding protein Orb2A is critical for the animals? ability to form and retain a memory. During the previous grant period, in our effort to decipher regulation of Orb2A aggregation, we discovered that the expression of Orb2A protein in the adult brain is controlled via a unique mechanism. The fully transcribed and polyadenylated Orb2A transcripts retain an intron and are stably expressed in the fly brain. The intron-retained Orb2A mRNA does not code for a protein, owing to multiple in frame stop codons. However, behavioral training that produces long-term but not short-term memory transiently increases the spliced protein-coding mRNA level. This splicing event, important for long-term memory, is mediated by a specific isoform of the splicing regulator Pasilla, psL. Intron retention (IR), a poorly studied form of alternative splicing, was thought to modulate gene expression primarily by degrading the mRNA, trapping it in the nucleus and thereby reducing protein expression. Our findings suggest that IR can be used in a novel way: to hold mRNA in a ?poised state?, which can allow spatial and temporal integration of various signals to acutely increase protein expression to regulate memory. These observations led us to ask how prevalent IR is in the adult Drosophila brain, and what is the molecular basis of IR in the brain and how experience interacts with the intron-retention mechanisms to control protein expression and memory. We have performed poly(A) RNA-sequencing from wild type fly brains. The analysis provided some surprising results. First, 8.9% of all detectable introns, corresponding to 3031 genes, show ? 25% retention in wildtype fly brain. These includes 55 genes with role in learning and memory. A recent genome-wide study revealed ~6% of fully transcribed and polyadenylated transcripts in the mouse brain also retains an intron. Remarkably, the mouse orthologues of some the Drosophila genes, including Orb2 orthologue CPEB2, also retains intron. Second, like Orb2, aggregate-prone prion-like proteins are significantly overrepresented in the intron retained genes. This includes a novel prion-like phosphatase we have recently identified. From adult brain, we have identified proteins that bind to a retained intron and interacts with psL. In the current proposal we intend to investigate how memory-related stimuli activate p38a, a putative kinase of psL, how behavioral stimuli effect psL interaction with its binding partners, what sequence motifs in a retained motif influence intron retention, how intron retention controls expression of aggregate-prone proteins and how perturbation of each of these processes effects animals ability to form and retain memory. These studies would provide insight into the IR-mediated control of protein expression, a novel gene regulatory mechanism, as well as the molecular basis of memory and the regulation of aggregate-prone proteins.

Public Health Relevance

At the molecular level, how memory formation is gated such that only some but not all experiences result in long- lasting memories, is an important unanswered question. The proposed study intends to investigate how intron retention controls expression of memory-related proteins to confer selectivity to long-lasting memory. We aim to determine how splicing machinery senses internal and external stimuli, identify trans-acting factors and regulatory sequence motifs, and the consequences of intron retention in memory formation and consolidation. These studies will provide the first detailed insight of intron retention in adult nervous system.

National Institute of Health (NIH)
National Institute of Mental Health (NIMH)
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Neurobiology of Learning and Memory Study Section (LAM)
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Driscoll, Jamie
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Stowers Institute for Medical Research
Kansas City
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Li, Liying; McGinnis, J P; Si, Kausik (2018) Translational Control by Prion-like Proteins. Trends Cell Biol 28:494-505
Gill, Jason; Park, Younshim; McGinnis, J P et al. (2017) Regulated Intron Removal Integrates Motivational State and Experience. Cell 169:836-848.e15
Li, Liying; Sanchez, Consuelo Perez; Slaughter, Brian D et al. (2016) A Putative Biochemical Engram of Long-Term Memory. Curr Biol 26:3143-3156
Khan, Mohammed Repon; Li, Liying; Pérez-Sánchez, Consuelo et al. (2015) Amyloidogenic Oligomerization Transforms Drosophila Orb2 from a Translation Repressor to an Activator. Cell 163:1468-83