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Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation
Published on: July 6, 2022
Expression and Characterization of Human Fragile X Mental Retardation Protein Isoforms and Interacting Proteins in
Jiang Zhang1, Guangli Wang2, Wei-Wu He2
1Department of Preventive Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.
Abstract:
Fragile X mental retardation protein is an mRNA-binding protein associated with phenotypic manifestations of fragile X syndrome, an X-linked disorder caused by mutation in the FMR1 gene that is the most common inherited cause of intellectual disability. Despite the well-studied genetic mechanism of the disease, the proteoforms of fragile X mental retardation protein have not been thoroughly characterized. Here, we report the expression and mass spectrometric characterization of human fragile X mental retardation protein. FMR1 cDNA clone was transfected into human HEK293 cells to express the full-length human fragile X mental retardation protein. Purified fragile X mental retardation protein was subjected to trypsin digestion and characterized by mass spectrometry. Results show 80.5% protein sequence coverage of fragile X mental retardation protein (Q06787, FMR1_HUMAN) including both the N- and C-terminal peptides, indicating successful expression of the full-length protein. Identified post-translational modifications include N-terminal acetylation, phosphorylation (Ser600), and methylation (Arg290, 471, and 474). In addition to the full-length fragile X mental retardation protein isoform (isoform 6), two endogenous fragile X mental retardation protein alternative splicing isoforms (isoforms 4 and 7), as well as fragile X mental retardation protein interacting proteins, were also identified in the co-purified samples, suggesting the interaction network of the human fragile X mental retardation protein. Quantification was performed at the peptide level, and this information provides important reference for the future development of a targeted assay for quantifying fragile X mental retardation protein in clinical samples. Collectively, this study provides the first comprehensive report of human fragile X mental retardation protein proteoforms and may help advance the mechanistic understanding of fragile X syndrome and related phenotypes associated with the FMR1 mutation.
Insights
This study characterizes human fragile X mental retardation protein (FXMRP) proteoforms using mass spectrometry. We identified post-translational modifications and alternative splicing isoforms, advancing understanding of fragile X syndrome.
Area of Science:
- Molecular Biology
- Proteomics
- Genetics
Background:
- Fragile X syndrome, the most common inherited intellectual disability, stems from FMR1 gene mutations.
- Fragile X mental retardation protein (FXMRP) is crucial, but its proteoforms remain poorly understood.
- Understanding FXMRP proteoforms is key to unraveling fragile X syndrome mechanisms.
Purpose of the Study:
- To express and characterize human FXMRP proteoforms.
- To identify post-translational modifications and alternative splicing variants of FXMRP.
- To provide a foundation for quantifying FXMRP in clinical settings.
Main Methods:
- Human HEK293 cells were transfected with FMR1 cDNA to express full-length FXMRP.
- Purified FXMRP underwent trypsin digestion and mass spectrometric analysis.
- Peptide-level quantification was performed for reference.
Main Results:
- Achieved 80.5% protein sequence coverage for full-length human FXMRP.
- Identified N-terminal acetylation, Ser600 phosphorylation, and methylation at Arg290, 471, 474.
- Detected alternative splicing isoforms (4 and 7) and interacting proteins.
Conclusions:
- This is the first comprehensive report on human FXMRP proteoforms.
- Findings offer insights into FXMRP's function and interaction network.
- The data supports future development of targeted FXMRP assays for fragile X syndrome.
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