Regulated T cell pre-mRNA splicing as genetic marker of T cell suppression

Includes abstract. Includes bibliographical references

Accumulation of splice variants and transcripts in response to PI3K inhibition in T cells

BACKGROUND: Measles virus (MV) causes T cell suppression by interference with phosphatidylinositol-3-kinase (PI3K) activation. We previously found that this interference affected the activity of splice regulatory proteins and a T cell inhibitory protein isoform was produced from an alternatively spliced pre-mRNA. Hypothesis Differentially regulated and alternatively splice variant transcripts accumulating in response to PI3K abrogation in T cells potentially encode proteins involved in T cell silencing. METHODS: To test this hypothesis at the cellular level, we performed a Human Exon 1.0 ST Array on RNAs isolated from T cells stimulated only or stimulated after PI3K inhibition. We developed a simple algorithm based on a splicing index to detect genes that undergo alternative splicing (AS) or are differentially regulated (RG) upon T cell suppression. RESULTS: Applying our algorithm to the data, 9% of the genes were assigned as AS, while only 3% were attributed to RG. Though there are overlaps, AS and RG genes differed with regard to functional regulation, and were found to be enriched in different functional groups. AS genes targeted extracellular matrix (ECM)-receptor interaction and focal adhesion pathways, while RG genes were mainly enriched in cytokine-receptor interaction and Jak-STAT. When combined, AS/RG dependent alterations targeted pathways essential for T cell receptor signaling, cytoskeletal dynamics and cell cycle entry. CONCLUSIONS: PI3K abrogation interferes with key T cell activation processes through both differential expression and alternative splicing, which together actively contribute to T cell suppression

Selected AS (<i>TRIM47</i>, <i>WRAP53</i>, <i>CHN1</i>; left) and RG (<i>DTL</i>, <i>SLFN5</i>; right) gene views showing probe set intensity plots (graphs) for each stimulated only (S, blue) or stimulated/inhibited (I, red) samples.

<p>Exons assigned with probe sets, location of primers within constant and AS regions and expected fragment lengths on RT-PCR analyses (which are exemplified each right to the corresponding graphs; one out of at least three independent experiments is shown) are indicated below the transcript profiles (A). Samples prepared as in A. were used for qPCR analyses with amplification levels for S (white bars) and I (black bars) being indicated. Data shown were obtained using a selected pair of RNAs (S and I), assays were performed in triplicate (B).</p

RG and AS class transcripts RT-PCR amplified for validation of class assignment performed on a representative number of independent RNAs for RGs.

<p>The frequencies of AS transcript analyses per variable (v) or constant (c) regions are indicated, and those matching AS criteria (differing in the AS region and regulated differently than the AS region within the C region) are indicated. Bold gene symbols mark genes for which, based on Gene annotation, as yet no alternatively spliced transcripts were described.</p

T cell receptor signaling pathway.

<p>Green indicates a down-regulated gene, red indicates an up-regulated gene. Spliced genes are marked in grey.</p

Figure 3

<p>Affected pathways based on SI genes (A) or genes commonly up-regulated or down-regulated (B). Red bars show the number of observed genes in the dataset, blue bars show the statistically expected number of genes, given the result to be random.</p

TNFSF/TNFR-SF related genes affected on PI3K disruption in T cells.

<p>TNFSF/TNFR-SF related genes affected on PI3K disruption in T cells.</p

Figure 2

<p>Overview of cell treatment, hybridization and prefiltering of the resulting data sets (A), selection of alternatively spliced genes (B) and selection of differentially expressed genes (C).</p