Structure and function analysis of Sam68 and hnRNP A1 synergy in the exclusion of exon 7 from SMN2 transcripts

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by the absence of a functional copy of the Survival of Motor Neuron 1 gene (SMN1). The nearly identical paralog, SMN2, cannot compensate for the loss of SMN1 because exon 7 is aberrantly skipped from most SMN2 transcripts, a process mediated by synergistic activities of Src‐associated during mitosis, 68 kDa (Sam68/KHDRBS1) and heterogeneous nuclear ribonucleoprotein (hnRNP) A1. This results in the production of a truncated, nonfunctional protein that is rapidly degraded. Here, we present several crystal structures of Sam68 RNA‐binding domain (RBD). Sam68‐RBD forms stable symmetric homodimers by antiparallel association of helices α3 from two monomers. However, the details of domain organization and the dimerization interface differ significantly from previously characterized homologs. We demonstrate that Sam68 and hnRNP A1 can simultaneously bind proximal motifs within the central region of SMN2 (ex7). Furthermore, we show that the RNA‐binding pockets of the two proteins are close to each other in their heterodimeric complex and identify contact residues using crosslinking‐mass spectrometry. We present a model of the ternary Sam68·SMN2 (ex7)·hnRNP A1 complex that reconciles all available information on SMN1/2 splicing. Our findings have important implications for the etiology of SMA and open new avenues for the design of novel therapeutics to treat splicing diseases.

[1]  E. Tizzano,et al.  The Importance of Digging into the Genetics of SMN Genes in the Therapeutic Scenario of Spinal Muscular Atrophy , 2021, International journal of molecular sciences.

[2]  P. Fontoura,et al.  Risdiplam-Treated Infants with Type 1 Spinal Muscular Atrophy versus Historical Controls. , 2021, The New England journal of medicine.

[3]  Jinan Wang,et al.  Recognition of single-stranded nucleic acids by small-molecule splicing modulators , 2021, Nucleic acids research.

[4]  R. Bordonné,et al.  Identification and structural analysis of the Schizosaccharomyces pombe SMN complex , 2021, Nucleic acids research.

[5]  A. Musarò,et al.  Sam68 splicing regulation contributes to motor unit establishment in the postnatal skeletal muscle , 2020, Life Science Alliance.

[6]  Robert J. Weatheritt,et al.  A Dynamic Splicing Program Ensures Proper Synaptic Connections in the Developing Cerebellum. , 2020, Cell reports.

[7]  B. Wirth,et al.  Twenty-Five Years of Spinal Muscular Atrophy Research: From Phenotype to Genotype to Therapy, and What Comes Next. , 2020, Annual review of genomics and human genetics.

[8]  P. Scheiffele,et al.  SAM68-Specific Splicing Is Required for Proper Selection of Alternative 3′ UTR Isoforms in the Nervous System , 2019, iScience.

[9]  Anna Knörlein,et al.  Structural basis of a small molecule targeting RNA for a specific splicing correction , 2019, Nature Chemical Biology.

[10]  A. Krainer,et al.  Antisense Oligonucleotide Therapies for Neurodegenerative Diseases. , 2019, Annual review of neuroscience.

[11]  B. Simon,et al.  Structural basis for terminal loop recognition and stimulation of pri-miRNA-18a processing by hnRNP A1 , 2018, Nature Communications.

[12]  F. Metzger,et al.  Targeting RNA structure in SMN2 reverses spinal muscular atrophy molecular phenotypes , 2018, Nature Communications.

[13]  P. Schultz,et al.  Mechanistic studies of a small-molecule modulator of SMN2 splicing , 2018, Proceedings of the National Academy of Sciences.

[14]  E. Aller,et al.  Correlation between SMA type and SMN2 copy number revisited: An analysis of 625 unrelated Spanish patients and a compilation of 2834 reported cases , 2018, Neuromuscular Disorders.

[15]  Adrian R. Krainer,et al.  Binding to SMN2 pre-mRNA-protein complex elicits specificity for small molecule splicing modifiers , 2017, Nature Communications.

[16]  Y. Hua,et al.  A-44G transition in SMN2 intron 6 protects patients with spinal muscular atrophy , 2017, Human molecular genetics.

[17]  F. Allain,et al.  Tandem hnRNP A1 RNA recognition motifs act in concert to repress the splicing of survival motor neuron exon 7 , 2017, eLife.

[18]  K. Talbot,et al.  The clinical landscape for SMA in a new therapeutic era , 2017, Gene Therapy.

[19]  Matthew R. Gazzara,et al.  A SLM2 Feedback Pathway Controls Cortical Network Activity and Mouse Behavior , 2016, Cell reports.

[20]  A. Krainer,et al.  Global identification of hnRNP A1 binding sites for SSO-based splicing modulation , 2016, BMC Biology.

[21]  N. Goshima,et al.  The Sam68 nuclear body is composed of two RNase-sensitive substructures joined by the adaptor HNRNPL , 2016, The Journal of cell biology.

[22]  S. Grellscheid,et al.  Structural basis of RNA recognition and dimerization by the STAR proteins T-STAR and Sam68 , 2016, Nature Communications.

[23]  A. Musarò,et al.  SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy , 2015, The Journal of cell biology.

[24]  Thomas M. Smith,et al.  SMN2 splice modulators enhance U1-pre-mRNA association and rescue SMA mice. , 2015, Nature chemical biology.

[25]  C. Lorson,et al.  Spinal muscular atrophy: a motor neuron disorder or a multi‐organ disease? , 2014, Journal of anatomy.

[26]  Brendan J. Frey,et al.  A compendium of RNA-binding motifs for decoding gene regulation , 2013, Nature.

[27]  Thomas Tuschl,et al.  Structure-function studies of STAR family Quaking proteins bound to their in vivo RNA target sites. , 2013, Genes & development.

[28]  G. Clowry,et al.  The Tissue-Specific RNA Binding Protein T-STAR Controls Regional Splicing Patterns of Neurexin Pre-mRNAs in the Brain , 2013, PLoS genetics.

[29]  Joonbae Seo,et al.  A Multi-Exon-Skipping Detection Assay Reveals Surprising Diversity of Splice Isoforms of Spinal Muscular Atrophy Genes , 2012, PloS one.

[30]  T. Bebee,et al.  Hypoxia is a modifier of SMN2 splicing and disease severity in a severe SMA mouse model. , 2012, Human molecular genetics.

[31]  D. Juan,et al.  Subtle mutations in the SMN1 gene in Chinese patients with SMA: p.Arg288Met mutation causing SMN1 transcript exclusion of exon7 , 2012, BMC Medical Genetics.

[32]  J. Valcárcel,et al.  hnRNP A1 proofreads 3' splice site recognition by U2AF. , 2012, Molecular cell.

[33]  Gene W. Yeo,et al.  Integrative genome‐wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins , 2012, Cell reports.

[34]  P. Scheiffele,et al.  SAM68 Regulates Neuronal Activity-Dependent Alternative Splicing of Neurexin-1 , 2011, Cell.

[35]  C. Sette,et al.  The RNA-binding protein Sam68 is a multifunctional player in human cancer. , 2011, Endocrine-related cancer.

[36]  T. Madl,et al.  Structural Basis for Homodimerization of the Src-associated during Mitosis, 68-kDa Protein (Sam68) Qua1 Domain*♦ , 2010, The Journal of Biological Chemistry.

[37]  S. Borrego,et al.  The c.859G>C variant in the SMN2 gene is associated with types II and III SMA and originates from a common ancestor , 2010, Journal of Medical Genetics.

[38]  M. Heiner,et al.  Proteomic identification of heterogeneous nuclear ribonucleoprotein L as a novel component of SLM/Sam68 Nuclear Bodies , 2009, BMC Cell Biology.

[39]  Y. Hua,et al.  A positive modifier of spinal muscular atrophy in the SMN2 gene. , 2009, American journal of human genetics.

[40]  S. Richard,et al.  The STAR RNA binding proteins GLD-1, QKI, SAM68 and SLM-2 bind bipartite RNA motifs , 2009, BMC Molecular Biology.

[41]  K. Fischbeck,et al.  Regulation of SMN Protein Stability , 2008, Molecular and Cellular Biology.

[42]  L. Regan,et al.  Structure and function of KH domains , 2008, The FEBS journal.

[43]  Lili Wan,et al.  SMN Deficiency Causes Tissue-Specific Perturbations in the Repertoire of snRNAs and Widespread Defects in Splicing , 2008, Cell.

[44]  C. Chalfant,et al.  The RNA-binding protein Sam68 modulates the alternative splicing of Bcl-x , 2007, The Journal of cell biology.

[45]  E. Androphy,et al.  Modulating role of RNA structure in alternative splicing of a critical exon in the spinal muscular atrophy genes , 2006, Nucleic acids research.

[46]  E. Androphy,et al.  An extended inhibitory context causes skipping of exon 7 of SMN2 in spinal muscular atrophy. , 2004, Biochemical and biophysical research communications.

[47]  J. Manley,et al.  A negative element in SMN2 exon 7 inhibits splicing in spinal muscular atrophy , 2003, Nature Genetics.

[48]  P. Herrlich,et al.  Signal-dependent regulation of splicing via phosphorylation of Sam68 , 2002, Nature.

[49]  J. Yong,et al.  Essential Role for the SMN Complex in the Specificity of snRNP Assembly , 2002, Science.

[50]  U. Monani,et al.  The survival motor neuron (SMN) protein: effect of exon loss and mutation on protein localization , 2000, Neurogenetics.

[51]  C. Lorson,et al.  An exonic enhancer is required for inclusion of an essential exon in the SMA-determining gene SMN. , 2000, Human molecular genetics.

[52]  J. McPherson,et al.  A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2. , 1999, Human molecular genetics.

[53]  K. Artzt,et al.  T-STAR/ETOILE: a novel relative of SAM68 that interacts with an RNA-binding protein implicated in spermatogenesis. , 1999, Human molecular genetics.

[54]  C. Lorson,et al.  A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[55]  G. Dreyfuss,et al.  The SMN–SIP1 Complex Has an Essential Role in Spliceosomal snRNP Biogenesis , 1997, Cell.

[56]  J. Weissenbach,et al.  Identification and characterization of a spinal muscular atrophy-determining gene , 1995, Cell.

[57]  C. Burd,et al.  RNA binding specificity of hnRNP A1: significance of hnRNP A1 high‐affinity binding sites in pre‐mRNA splicing. , 1994, The EMBO journal.

[58]  Ravindra N. Singh,et al.  Mechanism of Splicing Regulation of Spinal Muscular Atrophy Genes. , 2018, Advances in neurobiology.

[59]  F. Allain,et al.  Solution structure of the two RNA recognition motifs of hnRNP A1 using segmental isotope labeling: how the relative orientation between RRMs influences the nucleic acid binding topology , 2012, Journal of Biomolecular NMR.

[60]  S. Stamm,et al.  The splicing regulator Sam 68 binds to a novel exonic splicing silencer and functions in SMN 2 alternative splicing in spinal muscular atrophy , 2010 .

[61]  R. Touraine,et al.  A rare SMN2 variant in a previously unrecognized composite splicing regulatory element induces exon 7 inclusion and reduces the clinical severity of spinal muscular atrophy , 2010, Human mutation.

[62]  B. Davis,et al.  Sam68 RNA Binding Protein Is an In Vivo Substrate for Protein Arginine N-Methyltransferase 1 , 2003 .

[63]  F. Boisvert,et al.  A role for the GSG domain in localizing Sam68 to novel nuclear structures in cancer cell lines. , 1999, Molecular biology of the cell.