Cyclic stretch increases splicing noise rate in cultured human fibroblasts

Abstract

Background: Mechanical forces are known to alter the expression of genes, but it has so far not been reported whether they may influence the fidelity of nucleus-based processes. One experimental approach permitting to address this question is the application of cyclic stretch to cultured human fibroblasts. As a marker for the precision of nucleus-based processes, the number of errors that occur during co-transcriptional splicing can then be measured. This so-called splicing noise is found at low frequency in pre-mRNA splicing.

Findings: The amount of splicing noise was measured by RT-qPCR of seven exon skips from the test genes AATF, MAP3K11, NF1, PCGF2, POLR2A and RABAC1. In cells treated by altered uniaxial cyclic stretching for 18 h, a uniform and significant increase of splicing noise was found for all detectable exon skips.

Conclusion: Our data demonstrate that application of cyclic stretch to cultured fibroblasts correlates with a reduced transcriptional fidelity caused by increasing splicing noise.

Figure 1

Cold shock increases splicing noise rates in cultured fibroblasts. ΔCT of CT of the exon skip transcripts AATF-Δ3 (AATF), MAP3K11-Δ9 (MAP3K11), NF1-Δ39 (NF1S38), NF1-Δ38 (NF1S39), PCGF2-Δ10 (PCGF2), POLR2A-Δ23 (POLR2A), RABAC1-Δ4 (RABAC1) and of the CT of the wildtype transcripts in cDNA from untreated fibroblasts and fibroblasts treated with cold shock (differences in SD). Reduced ΔCT indicates an increased splicing noise rate.

Figure 2

Altered uniaxial cyclic stretching of fibroblasts doubles splicing noise rates. ΔCT of CT of the exon skip transcripts AATF-Δ3 (AATF), MAP3K11-Δ9 (MAP3K11), NF1-Δ39 (NF1S38), NF1-Δ38 (NF1S39) and RABAC1-Δ4 (RABAC1) to the CT of the wildtype transcripts in cDNA from fibroblasts cultured on PDMS (untreated) and treated by cyclic stretching (stretched) are shown.