Ratios of Four STAT3 Splice Variants in Human Eosinophils and Diffuse Large B Cell Lymphoma Cells

Abstract

Signal transducer and activator of transcription 3 (STAT3) is a key mediator of leukocyte differentiation and proliferation. The 3' end of STAT3 transcripts is subject to two alternative splicing events. One results in either full-length STAT3α or in STAT3β, which lacks part of the C-terminal transactivation domain. The other is at a tandem donor (5') splice site and results in the codon for Ser-701 being included (S) or excluded (ΔS). Despite the proximity of Ser-701 to the site of activating phosphorylation at Tyr-705, ΔS/S splicing has barely been studied. Sequencing of cDNA from purified eosinophils revealed the presence of four transcripts (S-α, ΔS-α, S-β, and ΔS-β) rather than the three reported in publically available databases from which ΔS-β is missing. To gain insight into regulation of the two alternative splicing events, we developed a quantitative(q) PCR protocol to compare transcript ratios in eosinophils in which STAT3 is upregulated by cytokines, activated B cell diffuse large B cell Lymphoma (DLBCL) cells in which STAT3 is dysregulated, and in germinal center B cell-like DLBCL cells in which it is not. With the exception of one line of activated B cell DLCBL cells, the four variants were found in roughly the same ratios despite differences in total levels of STAT3 transcripts. S-α was the most abundant, followed by S-β. ΔS-α and ΔS-β together comprised 15.6 ± 4.0 % (mean ± SD, n = 21) of the total. The percentage of STAT3β variants that were ΔS was 1.5-fold greater than of STAT3α variants that were ΔS. Inspection of Illumina's "BodyMap" RNA-Seq database revealed that the ΔS variant accounts for 10-26 % of STAT3 transcripts across 16 human tissues, with less variation than three other genes with the identical tandem donor splice site sequence. Thus, it seems likely that all cells contain the S-α, ΔS-α, S-β, and ΔS-β variants of STAT3.

Fig 1. Schematic of the four STAT3 splice variants.

The splice sites are near the 3' end of the transcript, so only the exons closest to this end are shown for simplicity. The non-coding 3’ UTR is shown in white, with grey representing different coding sequences as a result of alternative splicing. Phosphorylation sites present in the translated form are also shown on the transcript.

Fig 2. STAT3 S-β and ΔS-β splice variant transcripts are present in eosinophils.

PCR products were cloned into pET-Elmer vectors, amplified in E. coli and sequenced. The electrophoretogram depicts sequencing data from PCR amplification with a reverse primer specific for STAT3β. The amino acids encoded by the complementary sequence are shown below.

Fig 3. Validation of primer specificity.

The specificity of primers uniquely amplifying the four STAT3 splice variants was validated using mixed isoforms as template in PCR. An annealing temperature of 66°C was required to exclude S-α and S-β amplifications from the ΔS-α and ΔS-β reactions, respectively. DNA ladder is exACTGene 1 Kb Plus DNA Ladder (Fisher Scientific International Inc., Waltham, MA).

Fig 4. Pan-STAT3 primer quantification was compared to the summation of the four splice variants.

Quantification was performed for 18 eosinophil or DLBCL samples to validate the absolute qPCR protocol. Each point is plotted with its CV and represents the average of three replicates.

Fig 5. Splice variant ratios of eosinophil STAT3 change little when STAT3 transcription is induced.

(A) Relative quantification qPCR was used to determine fold changes in overall STAT3 levels in eosinophils over time under different conditions determined using GUSB as a reference gene. (B) Pie charts of STAT3 splice variants in untreated and treated eosinophils. Absolute quantification qPCR was used to determine proportions of STAT3 splice variants. Percentages of each splice variant are shown. Data represent averages determined from at least three reactions, with standard deviations shown for (A). IL3 = interleukin 3. TNFα = tumor necrosis factor α.

Fig 6. Pie charts of STAT3 splice variants in DLBCL.

Splice variant ratios in DLBCL differ minimally, except for RIVA. Absolute quantification qPCR was used to determine proportions of STAT3 splice variants in each cell line. Percentages of each splice variant are shown. Six samples are activated B cell DLBCL and two are germinal center B cell-like DLBCL. Data represent averages determined from at least three assays for duplicate reactions.

Fig 7. Analysis of “BodyMap” data.

(A) STAT3 PSI (percentage spliced in) values for at α/(α+β) vs S/(ΔS+S); with white blood cells denoted with a star. Our data based on qPCR are plotted with SEM. (B) Scatter Plot of PSI for 4 genes with GTAGTT donor splice sites. Data as mapped from Illumina BodyMap data across 16 tissues; within a minimum read count value of 15. Means and standard deviations are shown.