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

Mellert K, Uhl M, Högel J, Lamla M, Kemkemer R, Kaufmann D - Genes (Basel) (2011)

Bottom Line: In human pre-mRNA splicing, infrequent errors occur resulting in erroneous splice products as shown in a genome-wide approach.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Human Genetics, University of Ulm, Albert Einstein Allee 11, Ulm D 89070, Germany. kevin.mellert@uni-ulm.de.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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f2-genes-02-00562: 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.

Mentions: In NF1, the amount of the regular NF1 product was measured by qPCR using three different primer pairs binding to sequences flanking the skipped exons. The differences in the measurements of NF1 expression ranged within the deviation of multiple measurements expected from a single primer pair (Table 2). Therefore, in the following qPCR experiments, regular products were detected by using the WT38-39 primers only. Measurement of the four misspliced NF1 products resulted in low SDs (Figure 2), but the respective CT values differed in the cDNA of peripheral blood cells (Table 3) indicating that not every exon is erroneously spliced at the same frequency.


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

Mellert K, Uhl M, Högel J, Lamla M, Kemkemer R, Kaufmann D - Genes (Basel) (2011)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3927615&req=5

f2-genes-02-00562: 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.
Mentions: In NF1, the amount of the regular NF1 product was measured by qPCR using three different primer pairs binding to sequences flanking the skipped exons. The differences in the measurements of NF1 expression ranged within the deviation of multiple measurements expected from a single primer pair (Table 2). Therefore, in the following qPCR experiments, regular products were detected by using the WT38-39 primers only. Measurement of the four misspliced NF1 products resulted in low SDs (Figure 2), but the respective CT values differed in the cDNA of peripheral blood cells (Table 3) indicating that not every exon is erroneously spliced at the same frequency.

Bottom Line: In human pre-mRNA splicing, infrequent errors occur resulting in erroneous splice products as shown in a genome-wide approach.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Human Genetics, University of Ulm, Albert Einstein Allee 11, Ulm D 89070, Germany. kevin.mellert@uni-ulm.de.

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.

No MeSH data available.


Related in: MedlinePlus