title: Perturbation of chromatin structure globally affects localization and recruitment of splicing factors.
abstract: Chromatin structure is an important factor in the functional coupling between transcription and mRNA processing, not only by regulating alternative splicing events, but also by contributing to exon recognition during constitutive splicing. We observed that depolarization of neuroblastoma cell membrane potential, which triggers general histone acetylation and regulates alternative splicing, causes a concentration of SR proteins in nuclear speckles. This prompted us to analyze the effect of chromatin structure on splicing factor distribution and dynamics. Here, we show that induction of histone hyper-acetylation results in the accumulation in speckles of multiple splicing factors in different cell types. In addition, a similar effect is observed after depletion of the heterochromatic protein HP1α, associated with repressive chromatin. We used advanced imaging approaches to analyze in detail both the structural organization of the speckle compartment and nuclear distribution of splicing factors, as well as studying direct interactions between splicing factors and their association with chromatin in vivo. The results support a model where perturbation of normal chromatin structure decreases the recruitment efficiency of splicing factors to nascent RNAs, thus causing their accumulation in speckles, which buffer the amount of free molecules in the nucleoplasm. To test this, we analyzed the recruitment of the general splicing factor U2AF65 to nascent RNAs by iCLIP technique, as a way to monitor early spliceosome assembly. We demonstrate that indeed histone hyper-acetylation decreases recruitment of U2AF65 to bulk 3' splice sites, coincident with the change in its localization. In addition, prior to the maximum accumulation in speckles, ∼20% of genes already show a tendency to decreased binding, while U2AF65 seems to increase its binding to the speckle-located ncRNA MALAT1. All together, the combined imaging and biochemical approaches support a model where chromatin structure is essential for efficient co-transcriptional recruitment of general and regulatory splicing factors to pre-mRNA.
authors.affiliation: Laboratorio de Fisiología y Biología Molecular, Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Instituto de Fisiología, Biología Molecular y Neurociencias, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina.
authors.forename: Ignacio E
authors.initials: IE
authors.lastname: Schor
authors.forename: David
authors.initials: D
authors.lastname: Llères
authors.forename: Guillermo J
authors.initials: GJ
authors.lastname: Risso
authors.forename: Andrea
authors.initials: A
authors.lastname: Pawellek
authors.forename: Jernej
authors.initials: J
authors.lastname: Ule
authors.forename: Angus I
authors.initials: AI
authors.lastname: Lamond
authors.forename: Alberto R
authors.initials: AR
authors.lastname: Kornblihtt
mesh_terms: D000107:Acetylation; D017398:Alternative Splicing / Q000187:drug effects; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism*; D000090266:Chromobox Protein Homolog 5; D006657:Histones / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D008954:Models, Biological; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D022821:RNA Splice Sites; D012326:RNA Splicing / Q000187:drug effects / Q000502:physiology*; D062085:RNA, Long Noncoding / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C000715594:CBX5 protein, human; D002843:Chromatin; D006657:Histones; D006877:Hydroxamic Acids; D012322:RNA Precursors; D022821:RNA Splice Sites; D062085:RNA, Long Noncoding; D012261:Ribonucleoproteins; D000090266:Chromobox Protein Homolog 5; C012589:trichostatin A