Exon Junction Complexes Suppress Spurious Splice Sites to Safeguard Transcriptome Integrity.

Productive splicing of human precursor messenger RNAs (pre-mRNAs) requires the correct selection of authentic splice sites (SS) from the large pool of potential SS. Although SS consensus sequence and splicing regulatory proteins are known to influence SS usage, the mechanisms ensuring the effective suppression of cryptic SS are insufficiently explored. Here, we find that many aberrant exonic SS are efficiently silenced by the exon junction complex (EJC), a multi-protein complex that is deposited on spliced mRNA near the exon-exon junction. Upon depletion of EJC proteins, cryptic SS are de-repressed, leading to the mis-splicing of a broad set of mRNAs. Mechanistically, the EJC-mediated recruitment of the splicing regulator RNPS1 inhibits cryptic 5'SS usage, while the deposition of the EJC core directly masks reconstituted 3'SS, thereby precluding transcript disintegration. Thus, the EJC protects the transcriptome of mammalian cells from inadvertent loss of exonic sequences and safeguards the expression of intact, full-length mRNAs.

[1]  Helga Thorvaldsdóttir,et al.  Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration , 2012, Briefings Bioinform..

[2]  C. Béroud,et al.  Human Splicing Finder: an online bioinformatics tool to predict splicing signals , 2009, Nucleic acids research.

[3]  Thomas D. Schmittgen,et al.  Analyzing real-time PCR data by the comparative CT method , 2008, Nature Protocols.

[4]  E. Sakashita,et al.  Human RNPS1 and Its Associated Factors: a Versatile Alternative Pre-mRNA Splicing Regulator In Vivo , 2004, Molecular and Cellular Biology.

[5]  Zhen Wang,et al.  The exon junction complex as a node of post-transcriptional networks , 2015, Nature Reviews Molecular Cell Biology.

[6]  D. Durocher,et al.  High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities , 2015, Cell.

[7]  M. Hentze,et al.  Disassembly of Exon Junction Complexes by PYM , 2009, Cell.

[8]  L. Zou,et al.  PRP19 transforms into a sensor of RPA-ssDNA after DNA damage and drives ATR activation via a ubiquitin-mediated circuitry. , 2014, Molecular cell.

[9]  S. Salzberg,et al.  StringTie enables improved reconstruction of a transcriptome from RNA-seq reads , 2015, Nature Biotechnology.

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

[11]  Kunio Inoue,et al.  Splicing activator RNPS1 suppresses errors in pre-mRNA splicing: A key factor for mRNA quality control. , 2018, Biochemical and biophysical research communications.

[12]  J. Conboy,et al.  Intrasplicing coordinates alternative first exons with alternative splicing in the protein 4.1R gene , 2008, The EMBO journal.

[13]  R. Sachidanandam,et al.  The exon junction complex controls transposable element activity by ensuring faithful splicing of the piwi transcript , 2014, Genes & development.

[14]  Michael R. Green,et al.  The protein Sex-lethal antagonizes the splicing factor U2AF to regulate alternative splicing of transformer pre-mRNA , 1993, Nature.

[15]  Hanspeter Pfister,et al.  UpSet: Visualization of Intersecting Sets , 2014, IEEE Transactions on Visualization and Computer Graphics.

[16]  Z. Weng,et al.  The Cellular EJC Interactome Reveals Higher-Order mRNP Structure and an EJC-SR Protein Nexus , 2012, Cell.

[17]  P. Gendron,et al.  The Exon Junction Complex Controls the Splicing of mapk and Other Long Intron-Containing Transcripts in Drosophila , 2010, Cell.

[18]  Ravi Sachidanandam,et al.  Intrinsic differences between authentic and cryptic 5' splice sites. , 2003, Nucleic acids research.

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

[20]  J. Masters,et al.  Detection of Mycoplasma in cell cultures , 2010, Nature Protocols.

[21]  Christopher B. Burge,et al.  Maximum Entropy Modeling of Short Sequence Motifs with Applications to RNA Splicing Signals , 2004, J. Comput. Biol..

[22]  Panagiotis K. Papasaikas,et al.  The Spliceosome: The Ultimate RNA Chaperone and Sculptor. , 2016, Trends in biochemical sciences.

[23]  T. Maniatis,et al.  A systematic analysis of the factors that determine the strength of pre‐mRNA splicing enhancers , 1998, The EMBO journal.

[24]  D. Rio,et al.  Mechanisms and Regulation of Alternative Pre-mRNA Splicing. , 2015, Annual review of biochemistry.

[25]  T. Maniatis,et al.  Serine/arginine-rich protein-dependent suppression of exon skipping by exonic splicing enhancers. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Ross Smith,et al.  Functional diversity of the hnRNPs: past, present and perspectives. , 2010, The Biochemical journal.

[27]  Ana Cvejic,et al.  Inheritance of low-frequency regulatory SNPs and a rare null mutation in exon-junction complex subunit RBM8A causes TAR , 2012, Nature Genetics.

[28]  Larry N. Singh,et al.  U1 snRNP Determines mRNA Length and Regulates Isoform Expression , 2012, Cell.

[29]  Zefeng Wang,et al.  Haploinsufficiency for Core Exon Junction Complex Components Disrupts Embryonic Neurogenesis and Causes p53-Mediated Microcephaly , 2016, PLoS genetics.

[30]  T. Nilsen,et al.  Expansion of the eukaryotic proteome by alternative splicing , 2010, Nature.

[31]  Christopher W. J. Smith,et al.  Genome-Wide Association between Branch Point Properties and Alternative Splicing , 2010, PLoS Comput. Biol..

[32]  Guramrit Singh,et al.  The exon junction complex: a lifelong guardian of mRNA fate , 2017, Wiley interdisciplinary reviews. RNA.

[33]  Niels H. Gehring,et al.  Exon Junction Complexes: Supervising the Gene Expression Assembly Line. , 2016, Trends in genetics : TIG.

[34]  Niels H. Gehring,et al.  CWC22 connects pre-mRNA splicing and exon junction complex assembly. , 2012, Cell reports.

[35]  M. Blanchette,et al.  Human CWC22 escorts the helicase eIF4AIII to spliceosomes and promotes exon junction complex assembly , 2012, Nature Structural &Molecular Biology.

[36]  Brent S. Pedersen,et al.  Pybedtools: a flexible Python library for manipulating genomic datasets and annotations , 2011, Bioinform..

[37]  M. Hentze,et al.  The Hierarchy of Exon-Junction Complex Assembly by the Spliceosome Explains Key Features of Mammalian Nonsense-Mediated mRNA Decay , 2009, PLoS biology.

[38]  B. Séraphin,et al.  Structure of the Exon Junction Core Complex with a Trapped DEAD-Box ATPase Bound to RNA , 2006, Science.

[39]  Emanuele Buratti,et al.  DBASS3 and DBASS5: databases of aberrant 3′- and 5′-splice sites , 2010, Nucleic Acids Res..

[40]  Anne-Marie Alleaume,et al.  Exon Junction Complexes Show a Distributional Bias toward Alternatively Spliced mRNAs and against mRNAs Coding for Ribosomal Proteins , 2016, Cell reports.

[41]  Jean-Yves Roignant,et al.  Exon Junction Complex Subunits Are Required to Splice Drosophila MAP Kinase, a Large Heterochromatic Gene , 2010, Cell.

[42]  Nejc Haberman,et al.  Exon Junction Complex Shapes the Transcriptome by Repressing Recursive Splicing , 2018, Molecular cell.

[43]  Juan González-Vallinas,et al.  A new view of transcriptome complexity and regulation through the lens of local splicing variations , 2016, eLife.

[44]  J. Ebert,et al.  The Crystal Structure of the Exon Junction Complex Reveals How It Maintains a Stable Grip on mRNA , 2006, Cell.

[45]  H. Le Hir,et al.  Transcriptome-wide modulation of splicing by the exon junction complex , 2014, Genome Biology.

[46]  Jiuyong Xie,et al.  Diverse regulation of 3′ splice site usage , 2015, Cellular and Molecular Life Sciences.

[47]  J. Steitz,et al.  Human spliceosomal protein CWC22 plays a role in coupling splicing to exon junction complex deposition and nonsense-mediated decay , 2012, Proceedings of the National Academy of Sciences.

[48]  H. Le Hir,et al.  Exon Junction Complexes can have distinct functional flavours to regulate specific splicing events , 2018, Scientific Reports.

[49]  A. G. Murachelli,et al.  The structure of the ASAP core complex reveals the existence of a Pinin-containing PSAP complex , 2012, Nature Structural &Molecular Biology.

[50]  Niels H. Gehring,et al.  Studying the composition of mRNPs in vitro using splicing-competent cell extracts. , 2014, Methods.

[51]  Dominik Handler,et al.  The exon junction complex is required for definition and excision of neighboring introns in Drosophila , 2014, Genes & development.

[52]  David R. Kelley,et al.  Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks , 2012, Nature Protocols.

[53]  B. Blencowe,et al.  An atlas of alternative splicing profiles and functional associations reveals new regulatory programs and genes that simultaneously express multiple major isoforms , 2017, Genome Research.

[54]  Aaron R. Quinlan,et al.  BIOINFORMATICS APPLICATIONS NOTE , 2022 .

[55]  B. Blencowe,et al.  The RNA-binding profile of Acinus, a peripheral component of the exon junction complex, reveals its role in splicing regulation , 2016, RNA.

[56]  Gem Stapleton,et al.  Drawing Area-Proportional Euler Diagrams Representing Up To Three Sets , 2014, IEEE Transactions on Visualization and Computer Graphics.

[57]  Hedi Peterson,et al.  g:Profiler—a web server for functional interpretation of gene lists (2016 update) , 2016, Nucleic Acids Res..

[58]  L. Hurst,et al.  The evolution, impact and properties of exonic splice enhancers , 2013, Genome Biology.

[59]  Christopher R. Sibley,et al.  Lessons from non-canonical splicing , 2016, Nature Reviews Genetics.

[60]  Robert Castelo,et al.  Regulation of Fas alternative splicing by antagonistic effects of TIA-1 and PTB on exon definition. , 2005, Molecular cell.

[61]  J. Cáceres,et al.  The SR protein family of splicing factors: master regulators of gene expression. , 2009, The Biochemical journal.

[62]  David G Hendrickson,et al.  Differential analysis of gene regulation at transcript resolution with RNA-seq , 2012, Nature Biotechnology.

[63]  Michael Q. Zhang,et al.  Purification and characterization of human RNPS1: a general activator of pre‐mRNA splicing , 1999, The EMBO journal.

[64]  M. Passos-Bueno,et al.  A noncoding expansion in EIF4A3 causes Richieri-Costa-Pereira syndrome, a craniofacial disorder associated with limb defects. , 2014, American journal of human genetics.