Abstract
Dysregulated protein synthesis is a major underlying cause of many neurodevelopmental diseases including fragile X syndrome. In order to capture subtle but biologically significant differences in translation in these disorders, a robust technique is required. One powerful tool to study translational control is ribosome profiling, which is based on deep sequencing of mRNA fragments protected from ribonuclease (RNase) digestion by ribosomes. However, this approach has been mainly applied to rapidly dividing cells where translation is active and large amounts of starting material are readily available. The application of ribosome profiling to low-input brain tissue where translation is modest and gene expression changes between genotypes are expected to be small has not been carefully evaluated. Using hippocampal tissue from wide type and fragile X mental retardation 1 (Fmr1) knockout mice, we show that variable RNase digestion can lead to significant sample batch effects. We also establish GC content and ribosome footprint length as quality control metrics for RNase digestion. We performed RNase titration experiments for low-input samples to identify optimal conditions for this critical step that is often improperly conducted. Our data reveal that optimal RNase digestion is essential to ensure high quality and reproducibility of ribosome profiling for low-input brain tissue.
Highlights
Regulated protein synthesis in the synapto-dendritic compartment of neurons is essential to establish and maintain the brain circuitry that underlies higher cognitive function [1]
Repeat expansion in the 5 untranslated region (UTR) of fragile X mental retardation 1 (FMR1), which leads to epigenetic silencing and loss of its protein product, fragile X mental retardation protein (FMRP)
Sample batch effects dramatically compromise the power of ribosome profiling Ribosome profiling was initially established by Ingolia et al in yeast with RNase I, a nucleotide-indiscriminate endoribonuclease that preferentially hydrolyzes single-stranded RNA, to generate loaded 80S monomers [15]
Summary
Regulated protein synthesis in the synapto-dendritic compartment of neurons is essential to establish and maintain the brain circuitry that underlies higher cognitive function [1]. FMRP is a complex RNA binding protein that mediates gene expression at multiple levels, but acts most prominently as an inhibitor of translation [3]. FXS pathophysiologies may result from low abundance mRNAs that undergo large changes in translation or from the aggregation of small changes in translation of numerous mRNAs. in contrast to most cells, neurons are vulnerable to disruption of dosage and dynamics of RNAbinding proteins involved in RNA metabolism [7,8]. In contrast to most cells, neurons are vulnerable to disruption of dosage and dynamics of RNAbinding proteins involved in RNA metabolism [7,8] These unique features plus the fact that the brain is composed of multiple cell types pose significant challenges for reliably detecting differences in translation between normal versus disease conditions
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