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

Ignacio E. Schor,Andrea Pawellek,Angus I. Lamond,Guillermo J. Risso,Alberto R. Kornblihtt,David Llères,Jernej Ule,Laszlo Tora

doi:10.1371/journal.pone.0048084

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.

Highlights

Several molecular events related to the flow of the genetic information occur in the eukaryotic nucleus
We showed that an extracellular signal known to trigger histone acetylation and a drug that causes increased acetylation by inhibiting deacetylases activity have a similar effect on the nuclear distribution of these serine-arginine rich (SR) proteins, by promoting a conspicuous and quantitative concentration in nuclear speckles
This association with paraspeckles involved a minor fraction of U2AF65 (Fig. 5E and Fig. S9), suggesting that most of the splicing factor concentration in speckles is not related to the increase in expression of NEAT1 and the association with paraspeckles. It doesn’t exclude the possibility that both mechanisms are acting together and that, for example, the decrease of U2AF65 binding to the bulk of 39 splice sites at 6 h (Fig. 5A) could be mediated in part by sequestration of U2AF65 by NEAT1. It has been known for some time that HDAC inhibitors can affect alternative splicing choices, since the first report of trichostatin A (TSA) counteracting the effect of plasmid replication on alternative splicing of a fibronectin exon reporter [34]

Summary

Introduction

Several molecular events related to the flow of the genetic information occur in the eukaryotic nucleus. These events depend on the interplay between proteins and nucleic acids that do not act in isolation from other molecules, but generally associate in large complexes that harbor many molecular functions. In this way distinct steps in nuclear processes can be coupled with each other, thereby increasing efficiency. All steps of pre-mRNA processing, including capping, splicing and cleavage/poly-adenylation, are known to occur co-transcriptionally [5,6], to a different degree depending on the gene. In the case of splicing, organisms as divergent as mammals and yeasts show splicing factor recruitment as soon as their binding sites in the pre-mRNA have been transcribed [7,8,9]

Methods

Results

Conclusion