Fragile X mental retardation 1 (FMR1) encodes the RNA-binding protein, FMRP. Loss of FMRP is causative for Fragile X syndrome (FXS), the leading inherited cause of intellectual disability and most common monogenic form of autism. We recently defined for the first time, the global RNA targets of FMRP in human embryonic stem cells (hESCs) and in vitro derived excitatory cortical neurons. From these datasets, we identified roughly one-third of FMRP binding events on introns of pre-mRNA targets and detected a significant number of FMRP RNA targets transcribed from chromosome 21 (HSA21), the chromosome associated with Down syndrome. Many of these RNA targets were also bound as pre-mRNAs. While FMRP is most frequently studied in the context of translational regulation in the cytoplasm, emerging data point to additional functions of FMRP in transcriptional and post-transcriptional processes in the nucleus. However, the molecular mechanisms underlying the association between FMRP and pre-mRNAs remain unknown and are therefore the focus of this application. Given their disease relevance, we will specifically focus on the subset of FMRP pre-mRNA targets transcribed from HSA21. Indeed, our data support the novel hypothesis that the two most common genetic causes of intellectual disability, FXS and Down Syndrome (DS), converge on common molecular mechanisms. In Aim I, we selected a set of key HSA21-encoded FMRP targets to investigate the functional relevance of pre-mRNA binding events and downstream molecular consequences. We will now define the relevant FMRP domain mediating pre-mRNA binding, directly test the relevance of pre- mRNA binding for observed protein-level changes, and expand on the downstream molecular impact in patient cells. In Aim II, we will test the novel hypothesis that FMRP binds pre-mRNAs to play a direct inhibitory role in splicing, based in part on RNA-seq data showing differential transcript usage following FMRP loss. Specifically, we will examine how individual FMRP pre-mRNA binding sites regulate splicing products, assess the impact of FMRP loss on splicing kinetics and assess whether FMRP binding/splicing occurs co-transcriptionally. In Aim III, we will test the hypothesis that FMRP binding to chromatin underlies its association with pre-mRNAs using FMRP ChIP-seq, which would illuminate a novel mechanism of FMRP target recognition. We will further assess whether FMRP chromatin binding has independent effects on transcriptional output and probe the relationship between FMRP binding and known chromatin features. Collectively, this project will illuminate novel and fundamental aspects of FMRP biology in RNA processing, with the potential to impact our understanding of FXS and Down syndrome disease biology.
