RAN-Translation in Neurological Disease
A range of human neurological disorders occur due to nucleotide repeat expansions which can cause disease by protein gain-of-function, protein loss-of-function, or RNA gain-of-function. The FMR1 (Fragile X Messenger Ribonucleoprotein 1) gene is responsible for making FMRP; a protein found in the brain, ovaries and testes among many other tissues. FMRP is an RNA binding protein that is thought to control neuronal activity-dependent gene expression in post-synaptic neurons. In the absence of FMRP, translation becomes dysregulated and insufficient FMRP leads to dendrite pathology in the brain due to the protein’s involvement in the formation of dendritic spines. Fragile X-associated tremor ataxia syndrome (FXTAS) is a late-onset disorder and is a common inherited cause of dementia, tremor, and gait disorder and is caused by a moderately expanded CGG nucleotide repeat (55–200 repeats) in the 5′ UTR of FMR1. Much larger expansions (>200 repeats) of the same trinucleotide causes fragile X syndrome (FXS), an X-linked disorder caused by this CGG expansion in the FMR1 gene’s promoter region which subsequently becomes hypermethylated and thus silenced. FXS is the most common cause of autism as result of a single gene mutation. This article looks at three papers that utilise ribosome profiling to investigate the role of FMRP in the neurological diseases FXS and FXTAS. A basic overview of the relationship between the FMR1 gene, FMRP and both diseases can be seen in the figure below.
CGG Repeat-Associated Translation Mediates Neurodegeneration in Fragile X Tremor Ataxia Syndrome
Neuron, 2013, 78(3), pp.440-455.
Todd, P.K., Oh, S.Y., Krans, A., He, F., Sellier, C., Frazer, M., Renoux, A.J., Chen, K.C., Scaglione, K.M., Basrur, V. and Elenitoba-Johnson, K.
Repeat-associated non-AUG (RAN) translation involves unconventional initiation at disease-causing repeat expansions. In FXTAS patients, RAN translation occurs at the moderately expanded (55-200) CGG trinucleotide repeat and is associated with elevated FMR1 mRNA expression, neurodegeneration, and intranuclear neuronal inclusions. As RAN translation contributes to pathogenesis in many neurodegenerative disorders, determining its mechanism may benefit therapeutic development. The authors investigated the mechanism of inclusion formation in a Drosophila model of CGG repeat-mediated neurodegeneration. They did this by inserting the 5′ UTR containing 90 CGG repeats from an FXTAS patient upstream of the coding region for GFP in the Drosophila model. To test the theory that RAN translation occurs in FXTAS patients, they investigated pre-existing ribosome profiling data to assess whether the 5’ UTR of FMR1 mRNA is associated with translating ribosomes in human cell lines.
- RAN translation occurs in association with CGG repeats in FXTAS, a disease previously thought to result primarily from RNA-mediated toxicity.
- Production of FMRpolyG, a CGG RAN translation product in FXTAS, directly modulates CGG-associated pathology in two distinct model systems; mouse and drosophila.
The findings of this paper support the hypothesis in which RAN translation of an expanded polyglycine protein contributes to FXTAS disease pathogenesis, this suggests novel approaches toward therapeutic development in this and other neurodegenerative disorders with similar mechanisms.
FMRP links optimal codons to mRNA stability in neurons
Proceedings of the National Academy of Sciences, 2020, 117(48), pp.30400-30411.
Shu, H., Donnard, E., Liu, B., Jung, S., Wang, R. and Richter, J.D.,
Rarely is Autism spectrum disorder’s (ASD’s) aetiology so simple as a single gene mutation, yet that’s exactly what happens in FXS. Because it’s caused by a single gene, FXS is reasonably straightforward to model in mice by knocking out Fmr1, this is exactly what Shu et al. does. The authors modelled FXS using FMRP knockout (KO) mice and then used RNA metabolic profiling and ribosome profiling in FMRP-deficient mouse cortex slices. It is known from previous research that in FMRP-deficient mice, protein synthesis in the brain is elevated by up to 20%, indicating that FMRP represses translation. Using Ribo-seq and RNA-seq the authors investigated CPEB1 KO (CK) and FMRP/CPEB1 double knockout (dKO) mouse brain cortices in addition to the wild-type (WT) and FMRP KO (FK) samples.
- In the FK mouse brain cortex, steady-state mRNA levels are disrupted. RNA-seq in neurons identified that loss of FMRP results in mRNA instability in direct target substrates as well as other mRNAs with optimal codon bias.
- Over 50% of RNAs with differential ribosomal occupancy (DRO) in CPEB1 KO also had DROs in FMRP KO and were changed in the same direction (i.e., increased or decreased). DRO represents dysregulation of translational activity.
- In the dKO, CPEB1 depletion rescues FXS by rebalancing RNA homeostasis. Gene ontology analysis found that many of the up-regulated and rescued mRNAs have protein synthetic functions such as translation and ribosomal biogenesis, while the down-regulated and rescued mRNAs had transcription, synaptic transmission and chromatin remodelling functions.
It was shown that the main consequence of FMRP depletion in mouse brains is the uncoupling of codon bias from the RNA destruction machinery. This uncoupling may be a general mechanism that underlies FXS, and restoration of the RNA homeostasis could be a key to ameliorating the disorder.
FMRP control of ribosome translocation promotes chromatin modifications and alternative splicing of neuronal genes linked to autism
Cell Reports, 2020, 30(13), pp.4459-4472
Shah, S., Molinaro, G., Liu, B., Wang, R., Huber, K.M. and Richter, J.D.,
In their 2020 study, Shah et al. used ribosomal profiling to elucidate the role of FMRP in the hippocampus of Fmr1-/+ mice. The authors used several approaches to investigate FMRP regulation of mRNA expression in the mouse brain. They performed ribosomal profiling and RNA-seq at different time points after blocking initiation with homoharringtonine (HHT) in order to determine translocation rates. In addition, they also performed sucrose gradient analysis of the hippocampal-cortical slices treated with HHT for 30 minutes allowing majority of ribosomes to run off. RNA-seq of heavy fractions was then carried out to investigate which RNAs were elevated or reduced as a result of the HHT treatment. The same experiments were performed in both WT and Fmr1 KO slices.
- Using a combination of ribosome run-off and sucrose gradient centrifugation experiments, Shah et al. found that approximately 50 mRNAs, including the lysine methyltransferase SETD2, were associated with FMRP-stalled ribosomes.
- SETD2 is elevated in Fmr1 KO hippocampus. Chromatin immunoprecipitation sequencing (ChIP-seq) showed that increased SETD2 protein alters H3K36me3 marks in FMRP-deficient hippocampus.
- H3K36me3 has been correlated with alternative pre-mRNA splicing in mammals, as confirmed with the RNA-seq data from this study.
Alternative pre-mRNA processing is a common feature in autism. The authors deduce from their findings that perhaps mRNAs encoding splicing factors are improperly expressed in FXS and this leads to mis-splicing events. Overall, their observations show that while FXS is a disorder of improper translation that this is an oversimplification of the disease aetiology. The implications of this study is the revelation of the complexity of gene expression changes in FXS, which might offer opportunities for therapeutic intervention at multiple steps.
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