Aberrant Single Exon Skipping is not Altered by Age in Exons of NF1, RABAC1, AATF or PCGF2 in Human Blood Cells and Fibroblasts

Abstract

In human pre-mRNA splicing, infrequent errors occur resulting in erroneous splice products as shown in a genome-wide approach. One characteristic subgroup consists of products lacking one cassette exon. The noise in the splicing process, represented by those misspliced products, can be increased by cold shock treatment or by inhibiting the nonsense mediated decay. Here, we investigated whether the splicing noise frequency increases with age in vivo in peripheral bloods cells or in vitro in cultured and aged fibroblasts from healthy donors. Splicing noise frequency was measured for four erroneously skipped NF1 exons and one exon of RABAC1, AATF and PCGF2 by RT-qPCR. Measurements were validated in cultured fibroblasts treated with cold shock or puromycin. Intragenic but not interpersonal differences were detected in splicing noise frequencies in vivo in peripheral blood cells of 11 healthy donors (15 y-85 y) and in in vitro senescent fibroblasts from three further donors. No correlation to the age of the donors was found in the splicing noise frequencies. Our data demonstrates that splicing error frequencies are not altered by age in peripheral blood cells or in vitro aged fibroblasts in the tested exons of the four investigated genes, indicating a high importance of correct splicing in these proliferating aged cells.

Figure 1

(a) The specificity of NF1-Δ46 skip primers. (b) The specificity of RABAC1-Δ4 skip primers: The specificity of the primers to detect NF1-Δ46 or RABAC1-Δ4 erroneous splice products was tested on genomic DNA, cDNA of fibroblasts of a young donor (FP1) and 60mer oligonucleotides representing the wildtype sequence in standard PCR. The primer pairs did not generate products on genomic DNA and oligonucleotides after 60 cycles of amplification implicating that no products can be amplified as a reason of mispriming at sequences that match partly with the target sequence. To detect the length of the resulting products in the 3% agarose gel, a 25 bp marker was used. In lane 3 of panel A (water control) a weak primer band is visible.

Scheme I

General strategy of the PCR approach using the example of RABAC1-Δ4: After transcription of the gene, mRNA splicing leads either to a wildtype (WT) or an erroneously spliced product. The whole RNA was reverse transcribed to cDNA. The primers to detect the wildtype product are positioned in two consecutive exons (exon 3 and 4). The determining primers to detect the erroneously spliced product were designed as exon-boundary primer ranging over the speculative new exon-exon boundary after exon skipping (exon 3 and 5). To test the specificity of the skip primer pairs, gDNA and oligonucleotides representing the WT sequence at the skip primer binding position were used as template in a 60 cycle PCR. Determining skip primers can bind to the gDNA, oligonucleotide or wildtype cDNA only with 3–7 bases, so no product is amplified. Amplifications with cDNA as a template resulted in the clear products of the expected length. Absolute standard curves were performed by adding known amounts of synthetic templates representing the WT and skip primer sequences with a linker sequence in between. The amount of wildtype product molecules and skip product molecules in the measured cDNAs were calculated using the absolute standard curves.

Figure 2

qPCR to detect the NF1 wildtype and the transcript NF1-Δ52: The quantification of NF1 wildtype and of the misspliced transcripts in the cDNA of fibroblasts was performed by qPCR. Original amplification curves are represented. The amounts of mRNA molecules were calculated using an absolute standard curve. Splicing noise = the percentage of skip product in relation to the wildtype products. qPCR measurements were performed fourfold.

Figure 3

Cumulative population doublings of human fibroblasts of a 3 year old healthy donor (FP1): Cell doublings were estimated at the indicated times by counting the living cells. Fibroblasts of FP1 of passages 4 and 27, representing 4 or 45 weeks of cell culture, were investigated for splicing noise. Cells of passage 4 were defined as young, cells of passage 27 as old.