Loss of Tsc1 in cerebellar Purkinje cells induces transcriptional and translation changes in FMRP target transcripts
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Evaluation Summary:
The main strength of the manuscript is the data sets generated by the cell type-specific RNA-seq and TRAP-seq in cerebellar PCs that lack Tsc1. In addition, the bioinformatic analysis revealed several interesting findings, including the observation that FMRP target RNAs are reduced in the Tsc1 mutant PCs and that the translational efficiency of these RNAs is actually increased, likely through compensatory mechanisms.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Tuberous sclerosis complex (TSC) is a genetic disorder that is associated with multiple neurological manifestations. Previously, we demonstrated that Tsc1 loss in cerebellar Purkinje cells (PCs) can cause altered social behavior in mice. Here, we performed detailed transcriptional and translational analyses of Tsc1 -deficient PCs to understand the molecular alterations in these cells. We found that target transcripts of the Fragile X Mental Retardation Protein (FMRP) are reduced in mutant PCs with evidence of increased degradation. Surprisingly, we observed unchanged ribosomal binding for many of these genes using translating ribosome affinity purification. Finally, we found that multiple FMRP targets, including SHANK2, were reduced, suggesting that compensatory increases in ribosomal binding efficiency may be unable to overcome reduced transcript levels. These data further implicate dysfunction of FMRP and its targets in TSC and suggest that treatments aimed at restoring the function of these pathways may be beneficial.
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Reviewer #2 (Public Review):
In this manuscript, Dalal, Winden, and colleagues perform cell type-specific RNA-seq and TRAP-seq to analyze changes in total RNA levels and mRNA bound to L10/ribosomes in cerebellar Purkinje cells (PCs) that lack Tsc1, which when mutated gives rise to the developmental disorder tuberous sclerosis complex (TSC). These studies were motivated by previous studies by the Sahin laboratory that demonstrated that depletion of Tsc1 in cerebellar PCs alter social behavior in mice. The authors found that transcripts known to bind to RNA-binding protein fragile X mental retardation protein (FMRP) were reduced in the Tsc1 mutant PCs and subsequent bioinformatic analysis suggested that this was due to increased degradation of these RNAs. The TRAP-seq studies of the Tsc1 mutant PCs indicated that there was no change in …
Reviewer #2 (Public Review):
In this manuscript, Dalal, Winden, and colleagues perform cell type-specific RNA-seq and TRAP-seq to analyze changes in total RNA levels and mRNA bound to L10/ribosomes in cerebellar Purkinje cells (PCs) that lack Tsc1, which when mutated gives rise to the developmental disorder tuberous sclerosis complex (TSC). These studies were motivated by previous studies by the Sahin laboratory that demonstrated that depletion of Tsc1 in cerebellar PCs alter social behavior in mice. The authors found that transcripts known to bind to RNA-binding protein fragile X mental retardation protein (FMRP) were reduced in the Tsc1 mutant PCs and subsequent bioinformatic analysis suggested that this was due to increased degradation of these RNAs. The TRAP-seq studies of the Tsc1 mutant PCs indicated that there was no change in ribosomal binding for many of RNAs. Finally, that authors found that the FMRP target SHANK2, was reduced in PC synapses the Tsc1 mutant mice, suggesting that compensatory increases in ribosomal binding and translational efficiency is unable to overcome the reduction in transcript levels. The authors conclude their findings further implicate dysfunction of FMRP and its targets in TSC.
The main strength of the manuscript is the data sets generated by the cell type-specific RNA-seq and TRAP-seq in cerebellar PCs that lack Tsc1. In addition, the bioinformatic analysis revealed several interesting findings, including the observation that FMRP target RNAs are reduced in the Tsc1 mutant PCs, which may be due to degradation of these RNAs, and that the translational efficiency of these RNAs is actually increased, which may be a compensatory effect. The authors also observed that 5'TOP containing mRNAs showed increased translational efficiency in the Tsc1 mutant PCs, which is consistent with increased mTORC1 signaling. Finally, the authors show that the protein levels of SHANK2 are decreased in Tsc1 mutant PCs. Weaknesses include not examining the protein levels of additional proteins whose RNA levels are decreased and translation efficiency is increases and the lack of examination for protein levels for 5'TOP mRNAs that exhibit increased translational efficiency in Tsc1 mutant PCs. Given that the FMRP is thought to important for regulating the translation of long genes, it would be important to determine whether any of the differentially regulated genes in either the RNA-seq or TRAP-seq data sets correspond to the length of the gene. Otherwise, this an interesting manuscript that will be of interest to those studying translation, fragile X syndrome, and TSC.
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Reviewer #1 (Public Review):
This study reports that deletion of Tsc1 restricted to cerebellar Purkinje cells leads to decreased levels of mRNAs associated with FMRP targets although their ribosomal bindings are increased likely through compensatory mechanisms. However, protein levels of Shank2, one of the core FMRP targets, are decreased, suggesting that the compensatory increases in the ribosomal binding of FMRP target mRNAs do not rescue Shank2 protein expression. The analyses for transcriptional and translational processes were performed carefully and in a balanced manner, and the results are largely convincing and support the key conclusions. Considering the growing importance of mTOR signaling and cerebellar functions in ASD pathophysiology, the present work suggests that FMRP target transcripts are key mediators of Tsc1-related …
Reviewer #1 (Public Review):
This study reports that deletion of Tsc1 restricted to cerebellar Purkinje cells leads to decreased levels of mRNAs associated with FMRP targets although their ribosomal bindings are increased likely through compensatory mechanisms. However, protein levels of Shank2, one of the core FMRP targets, are decreased, suggesting that the compensatory increases in the ribosomal binding of FMRP target mRNAs do not rescue Shank2 protein expression. The analyses for transcriptional and translational processes were performed carefully and in a balanced manner, and the results are largely convincing and support the key conclusions. Considering the growing importance of mTOR signaling and cerebellar functions in ASD pathophysiology, the present work suggests that FMRP target transcripts are key mediators of Tsc1-related cerebellar dysfunctions, which is an important and timely contribution to the field.
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Evaluation Summary:
The main strength of the manuscript is the data sets generated by the cell type-specific RNA-seq and TRAP-seq in cerebellar PCs that lack Tsc1. In addition, the bioinformatic analysis revealed several interesting findings, including the observation that FMRP target RNAs are reduced in the Tsc1 mutant PCs and that the translational efficiency of these RNAs is actually increased, likely through compensatory mechanisms.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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