The Human RNA-Binding Proteome and Its Dynamics during Translational Arrest

Proteins and RNA functionally and physically intersect in multiple biological processes, however, currently no universal method is available to purify protein-RNA complexes. Here, we introduce XRNAX, a method for the generic purification of protein-crosslinked RNA, and demonstrate its versatility to study the composition and dynamics of protein-RNA interactions by various transcriptomic and proteomic approaches. We show that XRNAX captures all RNA biotypes and use this to characterize the sub-proteomes that interact with coding and non-coding RNAs (ncRNAs) and to identify hundreds of protein-RNA interfaces. Exploiting the quantitative nature of XRNAX, we observe drastic remodeling of the RNA-bound proteome during arsenite-induced stress, distinct from autophagy-related changes in the total proteome. In addition, we combine XRNAX with crosslinking immunoprecipitation sequencing (CLIP-seq) to validate the interaction of ncRNA with lamin B1 and EXOSC2. Thus, XRNAX is a resourceful approach to study structural and compositional aspects of protein-RNA interactions to address fundamental questions in RNA-biology.

[1]  L. J. Maher,et al.  p53 RNA interactions: new clues in an old mystery. , 2007, RNA.

[2]  B. Klaholz,et al.  Structure of the human 80S ribosome , 2015, Nature.

[3]  Peter Tompa,et al.  Polymer physics of intracellular phase transitions , 2015, Nature Physics.

[4]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[5]  Mikael Bodén,et al.  MEME Suite: tools for motif discovery and searching , 2009, Nucleic Acids Res..

[6]  R. Jackson,et al.  The mechanism of eukaryotic translation initiation and principles of its regulation , 2010, Nature Reviews Molecular Cell Biology.

[7]  Steven L Salzberg,et al.  Fast gapped-read alignment with Bowtie 2 , 2012, Nature Methods.

[8]  H. Ke,et al.  Beclin1 Controls the Levels of p53 by Regulating the Deubiquitination Activity of USP10 and USP13 , 2011, Cell.

[9]  M. Bohnsack,et al.  The roles of SSU processome components and surveillance factors in the initial processing of human ribosomal RNA , 2014, RNA.

[10]  E. Komives,et al.  RNAs interact with BRD4 to promote enhanced chromatin engagement and transcription activation , 2018, Nature Structural & Molecular Biology.

[11]  Paul Theodor Pyl,et al.  HTSeq—a Python framework to work with high-throughput sequencing data , 2014, bioRxiv.

[12]  R. Parker,et al.  Eukaryotic stress granules: the ins and outs of translation. , 2009, Molecular cell.

[13]  Thomas R. Gingeras,et al.  STAR: ultrafast universal RNA-seq aligner , 2013, Bioinform..

[14]  R. Parker,et al.  The Stress Granule Transcriptome Reveals Principles of mRNA Accumulation in Stress Granules. , 2017, Molecular cell.

[15]  Jeannie T. Lee,et al.  Chromosomes. A comprehensive Xist interactome reveals cohesin repulsion and an RNA-directed chromosome conformation. , 2015, Science.

[16]  Aaron T. L. Lun,et al.  csaw: a Bioconductor package for differential binding analysis of ChIP-seq data using sliding windows , 2015, Nucleic acids research.

[17]  M. Sohrmann,et al.  Mature ribosomes are selectively degraded upon starvation by an autophagy pathway requiring the Ubp3p/Bre5p ubiquitin protease , 2008, Nature Cell Biology.

[18]  M. Higuchi,et al.  Stress Granules Inhibit Apoptosis by Reducing Reactive Oxygen Species Production , 2012, Molecular and Cellular Biology.

[19]  Israel Steinfeld,et al.  BMC Bioinformatics BioMed Central , 2008 .

[20]  M. Mann,et al.  MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification , 2008, Nature Biotechnology.

[21]  J. Rinn,et al.  Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression , 2009, Proceedings of the National Academy of Sciences.

[22]  Jernej Ule,et al.  Advances in CLIP Technologies for Studies of Protein-RNA Interactions. , 2018, Molecular cell.

[23]  A. Chakraborty,et al.  An overview of pre-ribosomal RNA processing in eukaryotes , 2014, Wiley interdisciplinary reviews. RNA.

[24]  Anthony Barsic,et al.  ATPase-Modulated Stress Granules Contain a Diverse Proteome and Substructure , 2016, Cell.

[25]  Gene W. Yeo,et al.  Robust transcriptome-wide discovery of RNA binding protein binding sites with enhanced CLIP (eCLIP) , 2016, Nature Methods.

[26]  Norman E. Davey,et al.  Insights into RNA Biology from an Atlas of Mammalian mRNA-Binding Proteins , 2012, Cell.

[27]  Jeroen Krijgsveld,et al.  Comprehensive Identification of RNA-Binding Domains in Human Cells , 2016, Molecular cell.

[28]  Howard Y. Chang,et al.  Genome regulation by long noncoding RNAs. , 2012, Annual review of biochemistry.

[29]  D. Tollervey,et al.  Transcriptome-wide Analysis of Exosome Targets , 2012, Molecular cell.

[30]  P. Chomczyński,et al.  The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on , 2006, Nature Protocols.

[31]  Piero Carninci,et al.  Site-specific DICER and DROSHA RNA products control the DNA damage response , 2012, Nature.

[32]  M. Selbach,et al.  DDX54 regulates transcriptome dynamics during DNA damage response , 2017, Genome research.

[33]  Qiangfeng Cliff Zhang,et al.  Systematic Discovery of Xist RNA Binding Proteins , 2015, Cell.

[34]  M. Mann,et al.  Comparative Proteomic Analysis of Eleven Common Cell Lines Reveals Ubiquitous but Varying Expression of Most Proteins* , 2012, Molecular & Cellular Proteomics.

[35]  O. Kohany,et al.  Repbase Update, a database of repetitive elements in eukaryotic genomes , 2015, Mobile DNA.

[36]  Gene W. Yeo,et al.  Context-Dependent and Disease-Specific Diversity in Protein Interactions within Stress Granules , 2018, Cell.

[37]  Michael J. Sweredoski,et al.  The Xist lncRNA directly interacts with SHARP to silence transcription through HDAC3 , 2015, Nature.

[38]  S. Richard,et al.  Emerging Roles of Disordered Sequences in RNA-Binding Proteins. , 2015, Trends in biochemical sciences.

[39]  Jeroen Krijgsveld,et al.  Ultrasensitive proteome analysis using paramagnetic bead technology , 2014, Molecular systems biology.

[40]  Patrick B. F. O'Connor,et al.  Translation of 5′ leaders is pervasive in genes resistant to eIF2 repression , 2015, eLife.

[41]  Simone Sidoli,et al.  High-Resolution Mapping of RNA-Binding Regions in the Nuclear Proteome of Embryonic Stem Cells. , 2016, Molecular cell.

[42]  Oliver Kohlbacher,et al.  Photo-cross-linking and high-resolution mass spectrometry for assignment of RNA-binding sites in RNA-binding proteins , 2014, Nature Methods.

[43]  Nejc Haberman,et al.  Insights into the design and interpretation of iCLIP experiments , 2017, Genome Biology.

[44]  Alexey I Nesvizhskii,et al.  MSFragger: ultrafast and comprehensive peptide identification in shotgun proteomics , 2017, Nature Methods.

[45]  Charles Girardot,et al.  Je, a versatile suite to handle multiplexed NGS libraries with unique molecular identifiers , 2016, BMC Bioinformatics.

[46]  Richard Bonneau,et al.  The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. , 2012, Molecular cell.

[47]  J. Bishop,et al.  The expression of three abundance classes of messenger RNA in mouse tissues , 1976, Cell.

[48]  S. Gygi,et al.  Proteomic Analysis Identifies Ribosome Reduction as an Effective Proteotoxic Stress Response* , 2015, The Journal of Biological Chemistry.

[49]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[50]  Alexander Tretyakov,et al.  High-intensity UV laser ChIP-seq for the study of protein-DNA interactions in living cells , 2017, Nature Communications.

[51]  Katrin Eichelbaum,et al.  Selective enrichment of newly synthesized proteins for quantitative secretome analysis , 2012, Nature Biotechnology.

[52]  M. Selbach,et al.  Global quantification of mammalian gene expression control , 2011, Nature.

[53]  K. Neugebauer,et al.  How cells get the message: dynamic assembly and function of mRNA–protein complexes , 2013, Nature Reviews Genetics.

[54]  L. Tafforeau,et al.  The complexity of human ribosome biogenesis revealed by systematic nucleolar screening of Pre-rRNA processing factors. , 2013, Molecular cell.

[55]  C. Deng,et al.  BRCA1 Supports XIST RNA Concentration on the Inactive X Chromosome , 2002, Cell.

[56]  Robert B Darnell,et al.  HITS‐CLIP: panoramic views of protein–RNA regulation in living cells , 2010, Wiley interdisciplinary reviews. RNA.

[57]  G. Johnson,et al.  Degradation of protein translation machinery by amino acid starvation-induced macroautophagy , 2017, Autophagy.

[58]  Matthias W. Hentze,et al.  A brave new world of RNA-binding proteins , 2018, Nature Reviews Molecular Cell Biology.

[59]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..