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

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.

[1]  Xinyu Zhao,et al.  Regulatory discrimination of mRNAs by FMRP controls mouse adult neural stem cell differentiation , 2018, Proceedings of the National Academy of Sciences.

[2]  J. Rosenfeld,et al.  A Mild PUM1 Mutation Is Associated with Adult-Onset Ataxia, whereas Haploinsufficiency Causes Developmental Delay and Seizures , 2018, Cell.

[3]  E. Valjent,et al.  Cell Type-Specific mRNA Dysregulation in Hippocampal CA1 Pyramidal Neurons of the Fragile X Syndrome Mouse Model , 2017, Front. Mol. Neurosci..

[4]  N. Rajewsky,et al.  RNA localization is a key determinant of neurite-enriched proteome , 2017, Nature Communications.

[5]  Nicholas T Ingolia,et al.  Transcriptome-wide measurement of translation by ribosome profiling. , 2017, Methods.

[6]  J. Manley,et al.  RNA-binding proteins in neurodegeneration: mechanisms in aggregate , 2017, Genes & development.

[7]  Allen R. Buskirk,et al.  A ribosome profiling study of mRNA cleavage by the endonuclease RelE , 2016, Nucleic acids research.

[8]  R. Green,et al.  The DEAD-Box Protein Dhh1p Couples mRNA Decay and Translation by Monitoring Codon Optimality , 2016, Cell.

[9]  Gene W. Yeo,et al.  Genomic analysis of the molecular neuropathology of tuberous sclerosis using a human stem cell model , 2016, Genome Medicine.

[10]  Vadim N. Gladyshev,et al.  Ribonuclease selection for ribosome profiling , 2016, Nucleic acids research.

[11]  Nicholas T. Ingolia.  Ribosome Footprint Profiling of Translation throughout the Genome , 2016, Cell.

[12]  P. Worley,et al.  Selective Disruption of Metabotropic Glutamate Receptor 5-Homer Interactions Mimics Phenotypes of Fragile X Syndrome in Mice , 2016, The Journal of Neuroscience.

[13]  Rachel Green,et al.  Clarifying the Translational Pausing Landscape in Bacteria by Ribosome Profiling. , 2016, Cell reports.

[14]  E. Klann,et al.  Dysregulation of Mammalian Target of Rapamycin Signaling in Mouse Models of Autism , 2015, The Journal of Neuroscience.

[15]  Bong-Kiun Kaang,et al.  Multiple repressive mechanisms in the hippocampus during memory formation , 2015, Science.

[16]  Jeffrey A. Hussmann,et al.  Understanding Biases in Ribosome Profiling Experiments Reveals Signatures of Translation Dynamics in Yeast , 2015, bioRxiv.

[17]  Michael P Snyder,et al.  Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans , 2015, Genome research.

[18]  Jeffrey A. Hussmann,et al.  Improved ribosome-footprint and mRNA measurements provide insights into dynamics and regulation of yeast translation , 2015, bioRxiv.

[19]  R. Kooy,et al.  The GABAA Receptor as a Therapeutic Target for Neurodevelopmental Disorders , 2015, Neuron.

[20]  Pavel V. Baranov,et al.  Comparative survey of the relative impact of mRNA features on local ribosome profiling read density , 2015, Nature Communications.

[21]  James Taylor,et al.  Ribosome A and P sites revealed by length analysis of ribosome profiling data , 2015, Nucleic acids research.

[22]  M. Moore,et al.  An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments , 2014, Nucleic acids research.

[23]  Shu-Bing Qian,et al.  Quantitative profiling of initiating ribosomes in vivo , 2014, Nature Methods.

[24]  Hunter B. Fraser,et al.  Accounting for biases in riboprofiling data indicates a major role for proline in stalling translation , 2014, Genome research.

[25]  Sarah E. Jackson,et al.  Ribosome profiling reveals pervasive translation outside of annotated protein-coding genes. , 2014, Cell reports.

[26]  P. Canoll,et al.  Ribosome Profiling Reveals a Cell-Type-Specific Translational Landscape in Brain Tumors , 2014, The Journal of Neuroscience.

[27]  Huihao Zhou,et al.  Ribosome stalling induced by mutation of a CNS-specific tRNA causes neurodegeneration , 2014, Science.

[28]  S. Chattarji,et al.  Genetic and Acute CPEB Depletion Ameliorate Fragile X Pathophysiology , 2013, Nature Medicine.

[29]  Jonathan S. Weissman,et al.  rRNA:mRNA pairing alters the length and the symmetry of mRNA-protected fragments in ribosome profiling experiments , 2013, Bioinform..

[30]  J. Darnell,et al.  The translation of translational control by FMRP: therapeutic targets for FXS , 2013, Nature Neuroscience.

[31]  Shu-Bing Qian,et al.  Cotranslational response to proteotoxic stress by elongation pausing of ribosomes. , 2013, Molecular cell.

[32]  E. Klann,et al.  Exaggerated Translation Causes Synaptic and Behavioral Aberrations Associated with Autism , 2012, Nature.

[33]  B. Shen,et al.  Global mapping of translation initiation sites in mammalian cells at single-nucleotide resolution , 2012, Proceedings of the National Academy of Sciences.

[34]  Anna M. McGeachy,et al.  The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments , 2012, Nature Protocols.

[35]  V. Kim,et al.  Short structured RNAs with low GC content are selectively lost during extraction from a small number of cells. , 2012, Molecular cell.

[36]  Mark F Bear,et al.  The pathophysiology of fragile X (and what it teaches us about synapses). , 2012, Annual review of neuroscience.

[37]  Nicholas T. Ingolia,et al.  The translational landscape of mTOR signalling steers cancer initiation and metastasis , 2012, Nature.

[38]  J. Weissman,et al.  Selective Ribosome Profiling Reveals the Cotranslational Chaperone Action of Trigger Factor In Vivo , 2011, Cell.

[39]  Nicholas T. Ingolia,et al.  Ribosome Profiling of Mouse Embryonic Stem Cells Reveals the Complexity and Dynamics of Mammalian Proteomes , 2011, Cell.

[40]  J. Fak,et al.  FMRP Stalls Ribosomal Translocation on mRNAs Linked to Synaptic Function and Autism , 2011, Cell.

[41]  Nicholas T. Ingolia,et al.  Mammalian microRNAs predominantly act to decrease target mRNA levels , 2010, Nature.

[42]  Nicholas T. Ingolia,et al.  Genome-Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling , 2009, Science.

[43]  P. Greengard,et al.  A Translational Profiling Approach for the Molecular Characterization of CNS Cell Types , 2008, Cell.

[44]  Mark F. Bear,et al.  The Autistic Neuron: Troubled Translation? , 2008, Cell.

[45]  Bassem A. Hassan,et al.  Decreased expression of the GABAA receptor in fragile X syndrome , 2006, Brain Research.

[46]  Mark F. Bear,et al.  Altered synaptic plasticity in a mouse model of fragile X mental retardation , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[47]  M. Mazzocco,et al.  A developmental approach to understanding Fragile X syndrome in females , 2002, Microscopy research and technique.

[48]  Alexander Bartholomäus,et al.  Mapping the non-standardized biases of ribosome profiling , 2016, Biological chemistry.