Perturbation of Chromatin Structure Globally Affects Localization and Recruitment of Splicing Factors

<div class="autor_fcen" id="7966">Schor, I.E.</div>; Llères, D.; <div class="autor_fcen" id="7285">Risso, G.J.</div>; Pawellek, A.; Ule, J.; Lamond, A.I.; <div class="autor_fcen" id="4634">Kornblihtt, A.R.</div>
Publication Year
Published version
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. © 2012 Schor et al.
Fil:Schor, I.E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.
Fil:Risso, G.J. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.
Fil:Kornblihtt, A.R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.
PLoS ONE 2012;7(11)
enhanced green fluorescent protein
heterochromatin protein 1
heterochromatin protein 1alpha
histone H3
histone H4
messenger RNA
nuclear protein
protein SRSF1
protein SRSF2
trichostatin A
unclassified drug
untranslated RNA
cell nucleus
cellular distribution
chromatin structure
controlled study
gene control
gene function
histone acetylation
human cell
MALAT1 gene
molecular imaging
molecular model
NCAM gene
Neat1 gene
protein determination
protein function
protein RNA binding
regulatory mechanism
RNA analysis
RNA gene
RNA splicing
structure analysis
Alternative Splicing
Cell Line
Cell Nucleus
Hydroxamic Acids
Membrane Potentials
Models, Biological
Protein Binding
Protein Transport
RNA Precursors
RNA Splice Sites
RNA Splicing
RNA, Long Untranslated
Access level
Open access
Biblioteca Digital (UBA-FCEN)
Universidad Nacional de Buenos Aires. Facultad de Ciencias Exactas y Naturales
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