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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

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.
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f3-genes-02-00562: 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.

Mentions: Splicing fidelity may be influenced in vitro by replicative senescence. To test this, cultured fibroblasts from three donors (FP1, FP7 and FP8) were investigated at different passages (3–4 and 24–29), representing respectively 3–4 and more than 40 weeks of culture. The replicative senescence was shown by reduced population doubling (Figure 3), the altered morphology of the cells and the determination of the telomere length. A significant decrease in splicing noise frequency (p < 0.05; paired t-test) could be observed in NF1 exon 39 in in vitro aged fibroblasts of all three donors (FP1, FP7, FP8), but overall no significant change in splicing error frequency correlating with replicative senescence could be found (p = 0.39; sign test of the raw data; Table 5A). In addition, cultured fibroblasts (passages 5–6) from four donors differing in age (3–67 years) were investigated by pairwise testing a culture of a young and elderly donor (Table 5B). A comparison on the basis of four exons in two elderly and two young donors revealed slightly lower noise for the latter. This may however be attributable to chance (p = 0.52, ANOVA) and hence, no correlation between splicing noise frequency and the age of the donors could be detected under our culture conditions in vitro. The mean values ±SD per tested fibroblast pair over all tested NF1 exons point out that there is no significant difference between young and old fibroblasts/donors in our collective regarding the splicing error frequencies.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

f3-genes-02-00562: 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.
Mentions: Splicing fidelity may be influenced in vitro by replicative senescence. To test this, cultured fibroblasts from three donors (FP1, FP7 and FP8) were investigated at different passages (3–4 and 24–29), representing respectively 3–4 and more than 40 weeks of culture. The replicative senescence was shown by reduced population doubling (Figure 3), the altered morphology of the cells and the determination of the telomere length. A significant decrease in splicing noise frequency (p < 0.05; paired t-test) could be observed in NF1 exon 39 in in vitro aged fibroblasts of all three donors (FP1, FP7, FP8), but overall no significant change in splicing error frequency correlating with replicative senescence could be found (p = 0.39; sign test of the raw data; Table 5A). In addition, cultured fibroblasts (passages 5–6) from four donors differing in age (3–67 years) were investigated by pairwise testing a culture of a young and elderly donor (Table 5B). A comparison on the basis of four exons in two elderly and two young donors revealed slightly lower noise for the latter. This may however be attributable to chance (p = 0.52, ANOVA) and hence, no correlation between splicing noise frequency and the age of the donors could be detected under our culture conditions in vitro. The mean values ±SD per tested fibroblast pair over all tested NF1 exons point out that there is no significant difference between young and old fibroblasts/donors in our collective regarding the splicing error frequencies.

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