Biology subjects

D'Souza, M. N.

Publications and source records attributed to D'Souza, M. N..

3 recordsLinked to original sources

bioRxiv2024

Analysis of ribosomes from the Wild-type and FMR1 knockout human embryonic stem cells

Fragile X Messenger Ribonucleoprotein 1 (FMRP) is a multifunctional, multidomain RNA-binding protein whose loss causes Fragile X syndrome. It is also known to associate with ribosomes and modulate translation. In human embryonic stem cells (hESCs), knockout (KO) of the FMR1 gene results in significantly increased protein translation rates and alterations in the 2-O-methylation patterns of rRNA. To understand the structural underpinnings of the process, we performed electron cryomicroscopy analysis of ribosomes isolated from both wild-type (WT) and FMR1 KO hESCs that revealed a subpopulation of dormant ribosomes in the FMR1 KO cells, in addition to ribosomes with tRNAs. This dormant subpopulation, absent in the WT hESCs, is characterized by the binding of SERPINE-mRNA binding protein 1 in the mRNA tunnel and eukaryotic elongation factor 2 near the A-site, preventing translation. The presence of elevated protein translation in FMR1 KO cells, alongside a subpopulation of inactive ribosomes, suggests that FMRP can function as a translational brake. However, due to the high cost of ribosome recycling, the cell appears to adopt a strategy of maintaining a subset of dormant ribosomes. Additionally, we analysed the 2-O-methylation patterns in the 28S rRNA, in both the WT and FMR1 KO hESCs, identifying few potential differentially methylated sites. Thus, these findings provide insights into the mechanisms of ribosome dormancy in the absence of FMRP and lay the groundwork for understanding the role of rRNA methylation in translational regulation.

biochemistry↗

bioRxiv2024

Altering rRNA 2'O-methylation pattern during neuronal differentiation is regulated by FMRP

The Fragile X Messenger Ribonucleoprotein (FMRP) is a selective RNA-binding protein that localizes to the cytoplasm and the nucleus. The loss of FMRP results in Fragile X Syndrome (FXS), an Autism Spectrum Disorder. FMRP interacts with ribosomes and regulates the translation of mRNAs essential for neuronal development and synaptic plasticity. However, the biochemical nature of this translation regulation is unknown. Here we report that a key feature of FMRP-mediated translation regulation during neuronal differentiation is modulating the 2O-methylation of ribosomal RNA. 2O-methylation, facilitated by C/D box snoRNAs in the nucleus, is a major epitranscriptome mark on rRNA, essential for ribosome assembly and function. We found that FMRP influences a distinct rRNA 2O-Methylation pattern across neuronal differentiation. We show that in H9 ESCs, FMRP interacts with a selected set of C/D box snoRNA in the nucleus resulting in the generation of ribosomes with a distinct pattern of rRNA 2O-Methylation. This epitranscriptome pattern on rRNA undergoes a significant change during the differentiation of ESCs to neuronal precursors and cortical neurons. ESCs display maximum hypomethylated residues on rRNA, which is eventually reduced in neuronal precursors and post-mitotic cortical neurons and this is correlated to the change in global protein synthesis among the states of differentiation. Importantly, this gradual change in the 2O-methylation pattern during neuronal differentiation is altered in the absence of FMRP, which could impact neuronal development and contribute to dysregulated protein synthesis observed in Fragile X Syndrome. This also suggests the need for diversity in functional ribosomes during the early stages of development.

molecular biology↗

bioRxiv2021

Function of FMRP domains in regulating distinct roles of neuronal protein synthesis

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 and aids in their microtubule dependent transport and regulates their translation through its association with ribosomes. However, the biochemical role of individual domains of FMRP in forming neuronal granules and associating with microtubules and ribosomes is currently undefined. Here, we report that the C-terminus domain of FMRP is sufficient to bind to ribosomes as well as polysomes akin to the full-length protein. Furthermore, the C-terminus domain alone is essential and responsible for FMRP-mediated translation repression in neurons. However, FMRP-mediated puncta formation and microtubule association is favored by the synergistic combination of FMRP domains and not by individual domains. Interestingly, we show that the phosphorylation of hFMRP at Serine-500 is important in modulating the dynamics of translation by controlling ribosome/polysome association. This is a fundamental mechanism governing the size and number of FMRP puncta, which appear to contain actively translating ribosomes. Finally through the use of pathogenic mutations, we emphasize the hierarchy of the domains of FMRP in their contribution to translation regulation.

neuroscience↗