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.

The Peripheral Blood Eosinophil Proteome

“…For example, the proteomic data have already informed further study of the different splice isoforms of STAT3 from evidence of the STAT3 deletion 701 isoform in the proteomics data. 49 The sample preparation and further depletion of platelets indicate how studies of all types can be improved to limit impact from the platelets adhering to eosinophils. The publicly accessible data will provide a basis for eosinophil biologists to conduct future translational and clinical research on the similarities and differences in the proteome of eosinophils in different anatomic compartments and activation states in vivo; in different eosinophil-associated disorders such as asthma, eosinophilic gastrointestinal diseases, and hyper-eosinophilic syndrome; after different interventions and among different subjects; and on the possible associations of differences with disease activity, which are all currently areas of great interest.…”

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

“…Efficiencies between 83 and 95% were observed under the conditions described. The equation for specificity (Step 3.4) assumes equal efficiency, which is unlikely 25 , so the specificity is likely to be greater than specificity factor suggests.…”

“…This approach allows one to distinguish subtle differences in sequence and simultaneously measure splicing ratios at two alternative splice sites more than 50 nucleotides apart. Determining the ratios of the splicing events individually would not have yielded the remarkable finding that a co-splicing bias existed such that ΔSβ levels are higher than anticipated if uses of the two sites are randomly spliced 25 .…”

“…NOTE: KpnI and NheI sites were chosen to enable inserts to be ligated into a modified pET-28a vector with kanamycin resistance 25,26 . KpnI site had been added into the multiple cloning site.…”

“…This entails preparing a standard curve with known concentrations of the spliced transcript of interest, and ensuring PCR conditions optimize specificity 24 . As described, absolute and relative qPCR data for a particular gene can be merged to inform understanding of the gene's expression in a particular cell type, in this case STAT3 in variously stimulated eosinophils 25 .…”