RNA Control of Neural Function - Our work focuses on post-transcriptional control of neural function with emphasis on translational control of synaptic plasticity and learning and memory. We investigate the neurodevelopmental and neurodegenerative disorders that arise when this translation goes awry. We find that mis-regulated translation leads to changes in alternative splicing and RNA degradation, which in turn contribute to neuropathology. More specifically, our research is comprised of three distinct but complementary areas: (1) CPEB1 and cytoplasmic polyadenylation control of translation; (2) FMRP regulation of translation with emphasis on ribosome stalling and codon usage; (3) FMRP regulation of alternative splicing. CPEB1-regulated cytoplasmic polyadenylation governs translation in post-synaptic compartments, which in turn modifies synaptic strength, the underlying cellular basis of learning and memory. Molecular, electrophysiological, and behavioral experiments from our laboratory have demonstrated that CPEB1 regulates activity-dependent cytoplasmic polyadenylation-induced translation, which in turn modifies synaptic strength, and cognition. CPEB1 nucleates several proteins that promote poly(A) tail growth and removal and mediate translation initiation; they also regulate plasticity and animal behavior. Another RNA binding protein important for brain function is FMRP, the product of the Fragile X Syndrome gene FMR1. FMRP binds >1000 RNAs in the brain and regulates translation, primarily by stalling ribosome translocation on specific mRNAs. One such mRNA encodes the epigenetic factor SETD2, which catalyzes the chromatin mark H3K36me3. In FMRP-deficient mouse brain, SETD2 levels are elevated and the H3K36me3 chromatin landscape, which is principally located in gene bodies, is disrupted. H3K36me3 is linked to alternative pre-mRNA splicing and there is widespread mis-regulation of splicing in FMRP knockout (KO) mice. The main objectives our research going forward will address key unanswered questions regarding RNA regulation of neural function, primarily using mouse models: (a) CPEB1-deficiency rescues Fragile X pathophysiology in FMRP KO mice. Does this rescue involve ribosome stalling and/or polyadenylation? (b) CPEB1 also regulates 3’UTR length. What is the mechanism by which this occurs? (c) How does FMRP stall ribosomes on specific mRNAs? Does FMRP act as a molecular roadblock to ribosome transit and/or does FMRP interact with the ribosome? (d) How does FMRP regulate alternative splicing? Some of the mis-splicing events appear to involve H3K36me3, but these are in the minority. Does FMRP regulate the translation of mRNAs encoding splicing factors, and/or does FMRP, which is a shuttling protein, affect splicing directly? (e) How does FMRP employ codon optimality to regulate translation and RNA stability? We will address these issues by ribosome profiling, which we modified to distinguish between translocating and stalled ribosomes, and direct nanopore RNA sequencing, which yields poly(A) tail size at near-nucleotide resolution as well as RNA isoforms that arise by alternative splicing. We will deplete key regulatory factors from the mouse brain and assess synaptic function and animal behavior.
