SF3B1 knockdown and high but not low levels of an SF3B1 inhibitor decreases <i>HSF1</i> mRNA and protein levels.

<p>(A) qRT-PCR analysis of <i>HSF1</i> mRNA levels in cells that have been treated with either <i>HSF1</i> siRNA or <i>SF3B1</i> siRNA relative to nonsilencing siRNA control cells. (B) Western blot analysis of cells that have been treated with non-silencing control siRNA (NSC), <i>HSF1</i> siRNA, or <i>SF3B1</i> siRNA and probed with either anti-HSF1 or anti-ACTIN antibodies. (C) qRT-PCR analysis of <i>HSF1</i> mRNA levels in cells that have been treated with varying amounts of Pladienolide B relative to control cells. (D) Western blot analysis of cells that have been treated with varying amounts of Pladienolide B, with and without heat shock at 42° for 1 hour and probed with either anti-HSF1 or anti-ACTIN antibodies.</p

SF3B1 is a stress-sensitive splicing factor that regulates both HSF1 concentration and activity

<div><p>The heat shock response (HSR) is a well-conserved, cytoprotective stress response that activates the HSF1 transcription factor. During severe stress, cells inhibit mRNA splicing which also serves a cytoprotective function via inhibition of gene expression. Despite their functional interconnectedness, there have not been any previous reports of crosstalk between these two pathways. In a genetic screen, we identified SF3B1, a core component of the U2 snRNP subunit of the spliceosome, as a regulator of the heat shock response in <i>Caenorhabditis elegans</i>. Here, we show that this regulatory connection is conserved in cultured human cells and that there are at least two distinct pathways by which SF3B1 can regulate the HSR. First, inhibition of SF3B1 with moderate levels of Pladienolide B, a previously established small molecule inhibitor of SF3B1, affects the transcriptional activation of HSF1, the transcription factor that mediates the HSR. However, both higher levels of Pladienolide B and SF3B1 siRNA knockdown also change the concentration of HSF1, a form of HSR regulation that has not been previously documented during normal physiology but is observed in some forms of cancer. Intriguingly, mutations in SF3B1 have also been associated with several distinct types of cancer. Finally, we show that regulation of alternative splicing by SF3B1 is sensitive to temperature, providing a new mechanism by which temperature stress can remodel the transcriptome.</p></div

Depletion of SF3B1 inhibits the HSR.

<p>(A) qRT-PCR analysis of mRNA levels of two heat shock genes, <i>HSPA6</i> and <i>DNAJB1</i>, in cells that have been treated with either <i>HSF1</i> siRNA or <i>SF3B1</i> siRNA and subjected to a 1 hour heat shock at 42°. Data is shown relative to mRNA levels in heat shocked cells treated with a nonsilencing control siRNA. (B) Western blot analysis of cells treated with <i>SF3B1</i> siRNA or a nonsilencing control siRNA probed with both anti-SF3B1 and anti-ACTIN antibodies. Two biologically independent replicates for each sample are shown. (C) Western blot analysis of cells treated with <i>SF3B1</i> siRNA or a nonsilencing control siRNA and probed with anti-HSP70 or anti-ACTIN antibodies. Heat shocked cells were subjected to a 1 hour at 42° and then allowed to recover for 4 hours at 37° before harvesting. Induction levels of HSP70 were quantitated by densitometry from the blots and normalized to ACTIN. Fold induction upon heat shock is shown. (D) Diagram showing the qPCR primer locations for <i>HSPA6</i> and <i>DNAJB1</i>.</p

Model.

<p>(A) Model figure indicating the role of SF3B1 in regulation of the heat shock response. The central arrow indicates that SF3B1 regulates HSF1 concentration. The upper pathway indicates a second, indirect pathway where SF3B1 mediates regulation of HSF1 activity via an unknown factor X. The bottom pathway indicates heat shock stress conditions that both activate HSF1 activity and inhibit SF3B1. (B) Model figure demonstrating the relationship between the heat shock response, SF3B1 activity, and HSF1 levels.</p

Inhibition of SF3B1 affects HSF1 transcriptional activation.

<p>Quantitation of relative luminescence from cells expressing an HSF1-Gal4 chimera containing the HSF1 activation domain and the Gal4 DNA-binding domain exposed to varying levels of Pladienolide B. Luciferase activity was monitored from a firefly luciferase reporter with a Gal4 binding element that was co-transfected with the chimera. Luciferase activity was normalized to the no drug control and a VP16-Gal4 control. Dual luciferase with a general NanoLuc reporter was also measured to correct for differences in cell density and transfection efficiency. * indicates p-value < 0.05.</p

The SF3B1 inhibitor Pladienolide B inhibits the HSR in a dose-dependent manner.

<p>(A) qRT-PCR analysis of <i>HSPA6</i> mRNA levels in cells treated with varying levels of Pladienolide B (PB) and normalized to the no drug control. Cells were incubated with PB for 16 hours and then subjected to a 1 hour heat shock at 42° before harvest. (B) qRT-PCR analysis of an alternatively spliced variant of the <i>RBM5</i> transcript that is known to be sensitive to SF3B1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176382#pone.0176382.ref022" target="_blank">22</a>]. Cells were incubated with PB for 16 hours before harvest and normalized to the no drug control. (C) qRT-PCR analysis of the normal spliced transcript for <i>ACT1B</i> and <i>RBM5</i>. Cells were incubated with PB for 16 hours before harvest and normalized to the no drug control. (D) Diagram indicating primer locations for qPCR. * indicates p-value < 0.05.</p

SF3B1 activity is sensitive to temperature.

<p>qRT-PCR analysis of (A) an alternatively spliced variant of the <i>RBM5</i> transcript that is known to be regulated by SF3B1 and (B) the normal splice variant of <i>ACT1B</i> in cells incubated with varying levels of PB for 16 hours +/- heat shock at 42° for 1 hour before harvest. Data is normalized to the no drug, no HS control. * indicates p-value < 0.05.</p

List of oligonucleotide primers.

<p>List of oligonucleotide primers.</p

Positive Regulators of the HSR.

<p>Positive Regulators of the HSR.</p

Tissue-selective patterns for multiple HSR reporters.

<p>I = Intestine, M = Muscle, • = Induction, ○ = No Induction.</p