Pre-mRNA splicing is a determinant of nucleosome organization

Abstract

Chromatin organization affects alternative splicing and previous studies have shown that exons have increased nucleosome occupancy compared with their flanking introns. To determine whether alternative splicing affects chromatin organization we developed a system in which the alternative splicing pattern switched from inclusion to skipping as a function of time. Changes in nucleosome occupancy were correlated with the change in the splicing pattern. Surprisingly, strengthening of the 5' splice site or strengthening the base pairing of U1 snRNA with an internal exon abrogated the skipping of the internal exons and also affected chromatin organization. Over-expression of splicing regulatory proteins also affected the splicing pattern and changed nucleosome occupancy. A specific splicing inhibitor was used to show that splicing impacts nucleosome organization endogenously. The effect of splicing on the chromatin required a functional U1 snRNA base pairing with the 5' splice site, but U1 pairing was not essential for U1 snRNA enhancement of transcription. Overall, these results suggest that splicing can affect chromatin organization.

Figure 1. The shift in alternative splicing is linked to chromatin organization.

(A) Schematic diagram illustrating the exons (boxes) and introns (lines) in the IKAP19–23 minigene, which was inserted into pEGFP-C3 plasmid. The location of the different primers used is indicated. Drawing is not to scale. (B) The IKAP19–23 minigene was transfected into 293 cells. RNA samples were extracted 24, 48, or 72 hr following transfection. The splicing products were separated on a 2% agarose gel after RT-PCR reaction using appropriate primers. The PCR products were eluted and sequenced. (C) IKAP19–23 minigene was transfected into 293 cells. Nuclei were extracted 24 and 72 hr following transfection. Half of each sample was treated with MNase and half was untreated. Mononucleosomal DNA was extracted from an agarose gel and subjected to absolute QPCR analysis with primers that cover most of the minigene. Data are presented as DNA copy number and were normalized to transfection efficiency using primers for the GFP area of the plasmid using untreated samples.

Figure 2. Alternative splicing affects nucleosome occupancy.

(A) IKAP19–23 minigenes with different mutations that strengthened the 5′ss of each internal exon were transfected into 293 cells and splicing was evaluated. “Strong-ss” indicates a mutation that strengthened the splice site score . The positions of the 5′ss mutations for each minigene are indicated with an arrow in the lower part of the panel. (B) The IKAP19–23 minigene with a strong exon 20 5′ss was transfected into 293 cells, and nuclei were extracted 24 and 72 hr following transfection. Half of each sample was treated with MNase and half was untreated. Mononucleosomal DNA was extracted from an agarose gel and subjected to absolute QPCR analysis with primers that cover most of the minigene. Data are presented as DNA copy number and were normalized to transfection efficiency using primers for the GFP area of the plasmid using untreated samples. (C) Co-transfection of the IKAP19–23 minigene into 293 cells was performed with plasmids that express U1 snRNAs. Three different U1 snRNA plasmids were used (as shown in the lower part of the panel): wt U1, U1 with a mutation that strengthens its base pairing with exon 20 5′ss (U1 strong), and U1 with a mutation that weakens the base pairing with exon 20 5′ss (U1 weak). Watson-Crick base pairing is marked by dashed line. The position of the relevant mutation in U1 snRNA is indicated with an arrow. RNA samples were extracted 24, 48, or 72 hr following transfection. The splicing products were separated on a 2% agarose gel after RT-PCR. The PCR products were eluted and sequenced. (D) The IKAP19–23 minigene was co-transfected into 293 cells with either U1 wt, U1 strong or U1 weak plasmid. 72 hr after the transfection, nucleosome occupancy was determined using an MNase assay as above. All experiments were repeated independently three times, and the results shown are representative of an average experiment. QPCR experiments were performed in triplicate; results shown are mean values ± SD.

Figure 3. Alternative splicing affects nucleosome occupancy in endogenous genes.

(A) HeLa cells were treated with 10 nM meayamycin and RNA was extracted 24 hr later. Splicing products were separated on a 1.5% agarose gel after RT-PCR reaction using appropriate primers for the flanking the alternative exon (marked with an arrow). Ten endogenous genes were analyzed: BCL2-like 11 (apoptosis facilitator; BCL2L11); calcium channel, voltage-dependent, T type, alpha 1G subunit (CACNA1G); vacuolar protein sorting 26 homolog A (S. pombe) (VPS26A); calcium channel, voltage-dependent, T type, alpha 1H subunit (CACNA1H); cathepsin A (CTSA); polymerase delta interacting protein 3 (POLDIP3); KH domain containing, RNA binding, signal transduction associated 1 (KHDR); bromodomain containing 8 (BRD8); deoxyguanosine kinase, nuclear gene encoding mitochondrial protein (DGUOK); and proline-rich coiled coil 2B (PRRC2B). (B) DNA was extracted from HeLa nuclei 24 hr after meayamycin treatments. An MNase assay was then performed and the mononucleosomal DNA was subjected to absolute QPCR analysis on the alternative exon. Data are presented as DNA copy number.

Figure 4. Splicing affects RNAPII occupancy.

(A) At 72 hr following transfection with either the wt or strong exon 20 5′ss minigene, cells were collected and used for an RNAPII-ChIP analysis. The precipitated DNA fragments were subjected to QPCR. Enrichment values were normalized to the unbound fraction, to a non-specific IgG antibody, and to the GFP area of the plasmid. Results are presented as RNAPII fold change between strong exon 20 5′ss and the wt plasmid. (B) At 72 h following co-transfection of IKAP19–23 and U1 plasmids, RNAPII-ChIP was performed. All experiments were repeated independently three times, and the results shown are representative of an average experiment. QPCR experiments were amplified in triplicate; results shown are mean values ± SD.