title: Nuclear matrix-associated protein SMAR1 regulates alternative splicing via HDAC6-mediated deacetylation of Sam68.
abstract: Pre-mRNA splicing is a complex regulatory nexus modulated by various trans-factors and their posttranslational modifications to create a dynamic transcriptome through alternative splicing. Signal-induced phosphorylation and dephosphorylation of trans-factors are known to regulate alternative splicing. However, the role of other posttranslational modifications, such as deacetylation/acetylation, methylation, and ubiquitination, that could modulate alternative splicing in either a signal-dependent or -independent manner remain enigmatic. Here, we demonstrate that Scaffold/matrix-associated region-binding protein 1 (SMAR1) negatively regulates alternative splicing through histone deacetylase 6 (HDAC6)-mediated deacetylation of RNA-binding protein Sam68 (Src-associated substrate during mitosis of 68 kDa). SMAR1 is enriched in nuclear splicing speckles and associates with the snRNAs that are involved in splice site recognition. ERK-MAPK pathway that regulates alternative splicing facilitates ERK-1/2-mediated phosphorylation of SMAR1 at threonines 345 and 360 and localizes SMAR1 to the cytoplasm, preventing its interaction with Sam68. We showed that endogenously, SMAR1 through HDAC6 maintains Sam68 in a deacetylated state. However, knockdown or ERK-mediated phosphorylation of SMAR1 releases the inhibitory SMAR1-HDAC6-Sam68 complex, facilitating Sam68 acetylation and alternative splicing. Furthermore, loss of heterozygosity at the Chr.16q24.3 locus in breast cancer cells, wherein the human homolog of SMAR1 (BANP) has been mapped, enhances Sam68 acetylation and CD44 variant exon inclusion. In addition, tail-vein injections in mice with human breast cancer MCF-7 cells depleted for SMAR1 showed increased CD44 variant exon inclusion and concomitant metastatic propensity, confirming the functional role of SMAR1 in regulation of alternative splicing. Thus, our results reveal the complex molecular mechanism underlying SMAR1-mediated signal-dependent and -independent regulation of alternative splicing via Sam68 deacetylation.
authors.affiliation: Chromatin and Disease Biology Laboratory, National Centre for Cell Science, Pune 411007, India; Sprott Centre for Stem Cell Research, Ottawa Hospital Research Institute, Ottawa, ON, K1H 8L6, Canada;
authors.forename: Kiran Kumar
authors.initials: KK
authors.lastname: Nakka
authors.affiliation: Chromatin and Disease Biology Laboratory, National Centre for Cell Science, Pune 411007, India;
authors.forename: Nidhi
authors.initials: N
authors.lastname: Chaudhary
authors.affiliation: Chromatin and Disease Biology Laboratory, National Centre for Cell Science, Pune 411007, India;
authors.forename: Shruti
authors.initials: S
authors.lastname: Joshi
authors.affiliation: Department of Biophysics, Bose Institute, Kolkata 700054, India;
authors.forename: Jyotsna
authors.initials: J
authors.lastname: Bhat
authors.affiliation: Sprott Centre for Stem Cell Research, Ottawa Hospital Research Institute, Ottawa, ON, K1H 8L6, Canada;
authors.forename: Kulwant
authors.initials: K
authors.lastname: Singh
authors.affiliation: Department of Biophysics, Bose Institute, Kolkata 700054, India;
authors.forename: Subhrangsu
authors.initials: S
authors.lastname: Chatterjee
authors.affiliation: Advanced Center for Treatment, Education and Research, Tata Memorial Centre, Navi Mumbai 410210, India;
authors.forename: Renu
authors.initials: R
authors.lastname: Malhotra
authors.affiliation: Advanced Center for Treatment, Education and Research, Tata Memorial Centre, Navi Mumbai 410210, India;
authors.forename: Abhijit
authors.initials: A
authors.lastname: De
authors.affiliation: Cancer and Epigenetics Lab, National Centre for Cell Science, Pune 411007, India;
authors.forename: Manas Kumar
authors.initials: MK
authors.lastname: Santra
authors.affiliation: Sprott Centre for Stem Cell Research, Ottawa Hospital Research Institute, Ottawa, ON, K1H 8L6, Canada; Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, K1H 8L6, Canada.
authors.forename: F Jeffrey
authors.initials: FJ
authors.lastname: Dilworth
authors.affiliation: Chromatin and Disease Biology Laboratory, National Centre for Cell Science, Pune 411007, India; samit@nccs.res.in.
authors.forename: Samit
authors.initials: S
authors.lastname: Chattopadhyay
mesh_terms: D000107:Acetylation; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D017398:Alternative Splicing / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D055785:Gene Knockdown Techniques; D000073864:Histone Deacetylase 6; D006655:Histone Deacetylases / Q000378:metabolism*; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics; D020935:MAP Kinase Signaling System; D015530:Nuclear Matrix / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D021381:Protein Transport; D016601:RNA-Binding Proteins / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C417914:BANP protein, human; C497483:CD44 protein, human; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D018960:Hyaluronan Receptors; C489559:KHDRBS1 protein, human; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; C433811:HDAC6 protein, human; D000073864:Histone Deacetylase 6; D006655:Histone Deacetylases
keywords: HDAC6; SMAR1; Sam68; alternative splicing; deacetylation