Optimization of ribosome profiling using low-input brain tissue from fragile X syndrome model mice

AbstractLoss of the fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS). FMRP is widely thought to repress protein synthesis, but its translational targets and modes of control remain in dispute. We previously showed that genetic removal of p70 S6 kinase 1 (S6K1) corrects altered protein synthesis as well as synaptic and behavioral phenotypes in FXS mice. In this study, we examined the gene-specificity of altered mRNA translation in FXS and the mechanism of rescue with genetic reduction of S6K1 by carrying out ribosome profiling and RNA-Seq on cortical lysates from wild-type, FXS, S6K1 knockout, and double knockout mice. We observed reduced ribosome footprint abundance in the majority of differentially translated genes in the cortices of FXS mice. We used molecular assays to discover evidence that the reduction in ribosome footprint abundance reflects an increased rate of ribosome translocation, which is captured as a decrease in the number of translating ribosomes at steady state, and is normalized by inhibition of S6K1. We also found that genetic removal of S6K1 prevented a positive-to-negative gradation of alterations in translation efficiencies (RF/mRNA) with coding sequence length across mRNAs in FXS mouse cortices. Our findings reveal the identities of dysregulated mRNAs and a molecular mechanism by which reduction of S6K1 prevents altered translation in FXS.

Download Full-text

Finding novel distinctions between the sAPPα-mediated anabolic biochemical pathways in Autism Spectrum Disorder and Fragile X Syndrome plasma and brain tissue

Scientific Reports ◽

10.1038/srep26052 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 18

Author(s):

Balmiki Ray ◽

Deborah K. Sokol ◽

Bryan Maloney ◽

Debomoy K. Lahiri

Keyword(s):

Autism Spectrum Disorder ◽

Fragile X Syndrome ◽

Brain Tissue ◽

Fragile X ◽

Autism Spectrum ◽

Spectrum Disorder ◽

Biochemical Pathways

Download Full-text

Widespread Alterations in Translation Elongation in the Brain of Juvenile Fmr1 Knock-Out Mice

10.1101/319368 ◽

2018 ◽

Author(s):

Sohani Das Sharma ◽

Jordan B. Metz ◽

Hongyu Li ◽

Benjamin D. Hobson ◽

Nicholas Hornstein ◽

...

Keyword(s):

Fragile X Syndrome ◽

Translation Initiation ◽

Rna Binding ◽

Fragile X ◽

Ribosome Profiling ◽

Translation Elongation ◽

Knock Out ◽

Knock Out Mice ◽

Mrna Binding ◽

The Impact

SummaryFMRP is a polysome-associated RNA-binding protein encoded by Fmr1 that is lost in Fragile X syndrome. Increasing evidence suggests that FMRP regulates both translation initiation and elongation, but the gene-specificity of these effects is unclear. To elucidate the impact of Fmr1 loss on translation, we used ribosome profiling for genome-wide measurements of ribosomal occupancy and positioning in the cortex of 24 day-old Fmr1 knock-out mice. We found a remarkably coherent reduction in ribosome footprint abundance per mRNA for previously identified, high-affinity mRNA binding partners of FMRP, and an increase for terminal oligo-pyrimidine (TOP) motif-containing genes canonically controlled by mTOR-4EBP-eIF4E signaling. Amino acid motif- and gene-level analyses both showed a widespread reduction of translational pausing in Fmr1 knock-out mice. Our findings are consistent with a model of FMRP-mediated regulation of both translation initiation through eIF4E and elongation that is disrupted in Fragile X syndrome.

Download Full-text

Genetic removal of p70 S6K1 corrects coding sequence length-dependent alterations in mRNA translation in fragile X syndrome mice

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2001681118 ◽

2021 ◽

Vol 118 (18) ◽

pp. e2001681118

Author(s):

Sameer Aryal ◽

Francesco Longo ◽

Eric Klann

Keyword(s):

Protein Synthesis ◽

Fragile X Syndrome ◽

Messenger Rna ◽

Fragile X ◽

Mrna Translation ◽

Ribosome Profiling ◽

Sequence Length ◽

Double Knockout ◽

Coding Sequence ◽

Mental Retardation Protein

Loss of the fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS). FMRP is widely thought to repress protein synthesis, but its translational targets and modes of control remain in dispute. We previously showed that genetic removal of p70 S6 kinase 1 (S6K1) corrects altered protein synthesis as well as synaptic and behavioral phenotypes in FXS mice. In this study, we examined the gene specificity of altered messenger RNA (mRNA) translation in FXS and the mechanism of rescue with genetic reduction of S6K1 by carrying out ribosome profiling and RNA sequencing on cortical lysates from wild-type, FXS, S6K1 knockout, and double knockout mice. We observed reduced ribosome footprint (RF) abundance in the majority of differentially translated genes in the cortices of FXS mice. We used molecular assays to discover evidence that the reduction in RF abundance reflects an increased rate of ribosome translocation, which is captured as a decrease in the number of translating ribosomes at steady state and is normalized by inhibition of S6K1. We also found that genetic removal of S6K1 prevented a positive-to-negative gradation of alterations in translation efficiencies (RF/mRNA) with coding sequence length across mRNAs in FXS mouse cortices. Our findings reveal the identities of dysregulated mRNAs and a molecular mechanism by which reduction of S6K1 prevents altered translation in FXS.

Download Full-text