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Function of FMRP Domains in Regulating Distinct Roles of Neuronal Protein Synthesis

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Abstract

The Fragile-X Mental Retardation Protein (FMRP) is an RNA binding protein that regulates translation of mRNAs essential for synaptic development and plasticity. FMRP interacts with a specific set of mRNAs, aids in their microtubule-dependent transport and regulates their translation through its association with ribosomes. However, the biochemical role of FMRP’s domains in forming neuronal granules and associating with microtubules and ribosomes is currently undefined. We report that the C-terminus domain of FMRP is sufficient to bind to ribosomes akin to the full-length protein. Furthermore, the C-terminus domain alone is essential and responsible for FMRP-mediated neuronal translation repression. However, dendritic distribution of FMRP and its microtubule association is favored by the synergistic combination of FMRP domains rather than individual domains. Interestingly, we show that the phosphorylation of hFMRP at Serine-500 is important in modulating the dynamics of translation by controlling ribosome association. This is a fundamental mechanism governing the size and number of FMRP puncta that contain actively translating ribosomes. Finally through the use of pathogenic mutations, we emphasize the hierarchical contribution of FMRP’s domains in translation regulation.

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Data Availability

All data generated or analyzed during this study are included in this published article [and its supplementary information files.

Abbreviations

FMRP :

Fragile X mental retardation protein

RBP :

RNA binding protein

RPS6 :

Ribosomal protein S6

RPLP0 :

Ribosomal protein P0

RPL10 :

Ribosomal protein L10

NMDAR :

N-methyl-D-aspartate receptor

mGluR :

Metabotropic glutamate receptor

FXS :

Fragile X Syndrome

SNP :

Single nucleotide polymorphism

FUNCAT :

Fluorescent non-canonical amino-acid tagging

NLS :

Nuclear localization Signal

DMSO :

Di-methyl sulphoxide

ANOVA :

Analysis of variance

E.coli :

Escherichia coli

rRNA :

Ribosomal RNA

mRNA :

Messenger RNA

KH :

K-homology

WT :

Wild type

UTR :

Untranslated region

IPTG :

Isopropyl β-d-1-thiogalactopyranoside

BME :

Beta-mercaptoethanol

PBS :

Phosphate Buffer Saline

MMLV :

Moloney murine leukemia virus

ROI :

Region of interest

DTT :

Dithiotheritol

PTM :

Post-translational modification

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Acknowledgements

We thank Dr. Vinothkumar Kutti Ragunath (NCBS) for his useful comments and suggestions on the manuscript; Hitendra Negi (NCBS) for his help in our protein purification experiments; Sudhriti Ghosh Dastidar, CNS for his insights on FMRP and RPL10 puncta imaging and analysis; Central Imaging and Flow Cytometry Facility (CIFF) and Animal House Facility, NCBS-inStem; Members from the labs of Dr. Ravi Muddashetty, Dr .Dasaradhi Palakodeti, and Dr. Sumantra Chattarji (NCBS) for their invaluable suggestions and discussions.

Funding

The work was supported by the NeuroStem grant (BT/IN/Denmark/07/RSM/2015–2016) awarded to Dr. Ravi S Muddashetty and the DST Swarnajayanti Fellowship (DST/SJF/LSA-02/2015–16) awarded to Dr. Dasaradhi Palakodeti. Deepak Nair acknowledges the Department of Biotechnology-Indian Institute of science partnership (DBT-IISc) program and Indian Institute of Science under Institute of Eminence (IISc-IOE) program for the research and infrastructure support. Michelle Ninochka D’Souza was supported by the DST-Inspire Fellowship (DST/INSPIRE Fellowship/2018/IF180201) from the Department of Science and Technology (DST).

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M.N.D., S. Ramakrishna, S. Ravindran, D.N., and R.S.M.–designed the research; M.N.D., S. Ramakrishna, B.K.R., and V.J.–performed the research; L.Y. and D.N.–contributed to experimental tools; M.N.D., S. Ramakrishna, and D.N.–analyzed data; D.P. and R.S.M–provided funding; M.N.D–wrote the paper and M.N.D, S. Ramakrishna, S. Ravindran, B.K.R, V.J., L.Y., D.N, D.P., and R.S.M–reviewed versions of the manuscript.

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Correspondence to Ravi S. Muddashetty.

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D’Souza, M.N., Ramakrishna, S., Radhakrishna, B.K. et al. Function of FMRP Domains in Regulating Distinct Roles of Neuronal Protein Synthesis. Mol Neurobiol 59, 7370–7392 (2022). https://doi.org/10.1007/s12035-022-03049-1

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