A hypothetical model for the alternative splicing of <i>GLA</i> (IVS4 + 919G>A).

<p>A hypothetical model for the alternative splicing of <i>GLA</i> (IVS4 + 919G>A).</p

Modulation the alternative splicing of <i>GLA</i> (IVS4+919G>A) in Fabry disease

<div><p>While a base substitution in intron 4 of <i>GLA</i> (IVS4+919G>A) that causes aberrant alternative splicing resulting in Fabry disease has been reported, its molecular mechanism remains unclear. Here we reported that upon IVS4+919G>A transversion, H3K36me3 was enriched across the alternatively spliced region. PSIP1, an adapter of H3K36me3, together with Hsp70 and NONO were recruited and formed a complex with SF2/ASF and SRp20, which further promoted <i>GLA</i> splicing. Amiloride, a splicing regulator in cancer cells, could reverse aberrant histone modification patterns and disrupt the association of splicing complex with <i>GLA</i>. It could also reverse aberrant <i>GLA</i> splicing in a PP1-dependant manner. Our findings revealed the alternative splicing mechanism of <i>GLA</i> (IVS4+919G>A), and a potential treatment for this specific genetic type of Fabry disease by amiloride in the future.</p></div

Effects of RNA-associated proteins on the cryptic exon of <i>GLA</i> transcripts.

<p>(A-C) Virus-mediated shRNA knockdown of various genes as indicated. SMYD2 and NSD1 are histone methyltransferases which preferentially methylates Lys-36 of histone H3. (D) RNA-chromatin immunoprecipitations (RNA-ChIP) analysis on the cryptic exon area in Int4 of <i>GLA</i> was performed using antibodies against SF2/ASF and SRp20 with IgG as a control. (E) Co-immunoprecipitation results using the indicated antibodies for immunoprecipitation and analyzing by western blotting. FD cells, Fabry disease cells; NC: negative control; WB, western blot; SMYD2, SET and MYND domain containing 2; NSD1, nuclear receptor binding SET domain protein 1.</p

Effects of amiloride on the regulation of <i>GLA</i> (IVS4 + 919G>A) splicing.

<p>Cells were treated with different concentrations of amiloride for 24 hours and then harvested for RT-PCR analysis (A), or Western blot analysis (B,D). Actin and Histone H3 were used as internal standards. (C) The result of enzyme activity assay from FD cells after the treatment with or without amiloride for 24 hours. Data were presented as the mean ± standard deviation from three independent experiments. Asterisk represents significant difference (<i>p</i>-value < 0.05). (E) RT-PCR and Western blot results from cells pretreated with (+) or without (-) okadaic acid and then exposed to amiloride for 24 hours. FD cells, Fabry disease cells; Amil, amiloride; OA, okadaic acid.</p

Schematic representation of <i>GLA</i> transcripts.

<p>(A) Schematic illustration of <i>GLA</i> with positions of the biotin-labeled RNA probes for pull-down assays. (B) Putative regulatory motifs and binding sites for the splicing factors as determined by ESEfinder^a, Spliceaid2^b, Human Splicing Finder ^c, and our pull-down experiments*. ESS motifs and hnRNPA1 binding motifs were predicted to be disturbed upon IVS4 + 919G>A transversion. (C) Analyses of the RNA folding of <i>GLA</i> (IVS4 + 919G/A). The alternatively spliced 57 nucleotide sequence is highlighted in gray and the boxes indicate the alterations in RNA folding.</p

Effects of proteins associated with the cryptic exon area in Int4 of <i>GLA</i>.

<p>(A) Schematic illustration of <i>GLA</i> with positions of the biotin-labelled DNA probes for pull-down assays. (B) ChIP analysis on the cryptic exon area in Int4 of <i>GLA</i> was performed using antibodies against HSP70, NONO, and H3K36me3 with IgG as a control. (C) Co-immunoprecipitation results using anti- HSP70 or anti-NONO antibody for immunoprecipitation and analyzing by western blotting. Nonimmune IgG was used as negative control. (D) Fabry disease cells were infected with lentiviruses expressing shRNAs targeting <i>HSP70</i> or <i>NONO</i>, or treated with 4β-hydroxywithanolide E (4HWE). Messenger RNA was extracted after 48 hours infection or 24 hours 4HWE treatment followed by RT-PCR analysis. WB, western blot; NC: negative control.</p

Alterations in histone modification patterns after amiloride treatment.

<p>ChIP assays were performed with antibodies to the indicated histone modifications on the cryptic exon area in Int4 of <i>GLA</i> in normal cells or in FD cells treated with or without amiloride. Results were expressed as a fraction of histone H3 after normalization to input values and presented as a mean values ± standard deviation from three independent experiments. Asterisk represents significant difference (<i>p</i>-value < 0.05). FD cells, Fabry disease cells; Amil, amiloride.</p

Alterations of histone modifications in FD cells.

<p>(A) Schematic representation of position and sequence of primer/probe sets used for real-time PCR. (B) ChIP assays were performed with antibodies to the indicated histone modifications across the alternatively spliced region (exon 4-intron 4-exon 5) of <i>GLA</i> in normal cells and FD cells. Results were expressed as a fraction of histone H3 after normalization to input values and presented as a mean values ± standard deviation from at least three independent experiments. Asterisk represents significant difference (<i>p</i>-value < 0.05). (C) Fabry disease cells were treated with two different histone acetyltransferase (HAT) inhibitors, C646 and HAT inhibitor VII, for 24 hours. The effects of histone acetylation on alternative splicing of <i>GLA</i> were detected by RT-PCR.</p

Alternative splicing of <i>GLA</i> (IVS4 + 919G>A).

<p>(A) Schematic representation of <i>GLA</i>. Uppercase letters indicate the exonic sequences, whereas lowercase letters indicate the intronic sequences. The encoded amino acids are depicted in single-letter code. The invariant AG and GT dinucleotides (the 3’ and 5’ splice sites) are shown in boldface type. The alternatively spliced 57 nucleotide sequence is enclosed in the box with italics letters. (B) Messenger RNA was extracted and detected by RT-PCR for alternative splicing of <i>GLA</i> (IVS4 + 919G>A). The splicing variants and their expected PCR products using the primers indicated by arrowheads are illustrated on the right column. (C) Aliquots containing 20 μg of whole cell lysates was subjected to SDS-PAGE followed by immunoblot analysis using an anti-GLA antibody. Actin was shown as internal standard. (D) The result of enzyme activity assay from lymphoid cell lines of health person and FD patient. Data were presented as the mean ± standard deviation from three independent experiments. Asterisk represents significant difference (<i>p</i>-value < 0.05). N, normal cells; FD, Fabry disease cells.</p

Additional file 2: of Long noncoding RNA TUG1 is downregulated in non-small cell lung cancer and can regulate CELF1 on binding to PRC2

RIP and DNA ChIP. (DOCX 15 kb