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Integrative omics indicate FMRP sequesters mRNA from translation and deadenylation in human neuronal cells
Tatsuaki Kurosaki1, Shuhei Mitsutomi2, Alexander Hewko1
1Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA; Center for RNA Biology, University of Rochester, Rochester, NY 14642, USA.
Abstract:
How fragile X syndrome protein (FMRP) binds mRNAs and regulates mRNA metabolism remains unclear. Our previous work using human neuronal cells focused on mRNAs targeted for nonsense-mediated mRNA decay (NMD), which we showed are generally bound by FMRP and destabilized upon FMRP loss. Here, we identify >400 high-confidence FMRP-bound mRNAs, only ∼35% of which are NMD targets. Integrative transcriptomics together with SILAC-LC-MS/MS reveal that FMRP loss generally results in mRNA destabilization and more protein produced per FMRP target. We use our established RIP-seq technology to show that FMRP footprints are independent of protein-coding potential, target GC-rich and structured sequences, and are densest in 5' UTRs. Regardless of where within an mRNA FMRP binds, we find that FMRP protects mRNAs from deadenylation and directly binds the cytoplasmic poly(A)-binding protein. Our results reveal how FMRP sequesters polyadenylated mRNAs into stabilized and translationally repressed complexes, whose regulation is critical for neurogenesis and synaptic plasticity.
Insights
Fragile X syndrome protein (FMRP) binds mRNAs, protecting them from decay and translation. Loss of FMRP leads to mRNA destabilization and increased protein production, impacting neurogenesis and synaptic plasticity.
Area of Science:
- Molecular Biology
- Neuroscience
- Genetics
Background:
- The function of fragile X syndrome protein (FMRP) in mRNA binding and metabolism is not fully understood.
- Previous studies indicated FMRP targets mRNAs for nonsense-mediated mRNA decay (NMD).
Purpose of the Study:
- To identify FMRP-bound mRNAs beyond NMD targets.
- To elucidate the mechanisms by which FMRP regulates mRNA stability and translation.
Main Methods:
- RNA immunoprecipitation sequencing (RIP-seq) to identify FMRP-bound mRNAs.
- Integrative transcriptomics and SILAC-LC-MS/MS to assess mRNA and protein changes upon FMRP loss.
- Analysis of FMRP binding site characteristics.
Main Results:
- Over 400 high-confidence FMRP-bound mRNAs were identified, with only ~35% being NMD targets.
- Loss of FMRP generally leads to mRNA destabilization and increased protein production.
- FMRP binds GC-rich, structured sequences, primarily in 5' UTRs, protecting mRNAs from deadenylation by interacting with poly(A)-binding protein.
Conclusions:
- FMRP sequesters polyadenylated mRNAs into stabilized, translationally repressed complexes.
- This regulation by FMRP is crucial for neurogenesis and synaptic plasticity.
- FMRP's role extends beyond NMD, impacting global mRNA metabolism.
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