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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

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.
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f4-genes-02-00562: 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.

Mentions: Splicing noise frequencies were measured by RT-qPCR of the regular NF1, PCGF2, RABAC1 and AATF transcripts (wildtype products) in relation to the products without the skipped exons (NF1-Δ38, NF1-Δ39, NF1-Δ46, NF1-Δ52, PCGF2-Δ10, RABAC1-Δ4 and AATF-Δ3). The investigated skipped exons differ in length, in upstream and downstream intron length, splice site strengths (calculated with SplicePort [19]) and in generating a frameshift as consequence of the exon skip (Table 1). The specificity of the skip primers was tested by PCR (60 cycles) on genomic DNA and wildtype 60mer oligonucleotides. The oligonucleotides represent the wildtype sequence at the position of skip primer binding and enable detection of mispriming of the skip primers (Scheme I). The method was tested using published UBA52 and RPL23 primers known to misanneal at low frequency [1]. PCRs using those primers resulted in clear bands on gDNA. In contrast, in experiments using the skip primers used in our measurements, no products were found in PCR on gDNA or oligonucleotides, whereas PCRs on cDNA of fibroblasts resulted in the expected products (Figure 1). This implicates that misannealing with these primers may occur at even lower frequency than that published for the UBA52 and RPL23 primers (found to misanneal <1:1000).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

f4-genes-02-00562: 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.
Mentions: Splicing noise frequencies were measured by RT-qPCR of the regular NF1, PCGF2, RABAC1 and AATF transcripts (wildtype products) in relation to the products without the skipped exons (NF1-Δ38, NF1-Δ39, NF1-Δ46, NF1-Δ52, PCGF2-Δ10, RABAC1-Δ4 and AATF-Δ3). The investigated skipped exons differ in length, in upstream and downstream intron length, splice site strengths (calculated with SplicePort [19]) and in generating a frameshift as consequence of the exon skip (Table 1). The specificity of the skip primers was tested by PCR (60 cycles) on genomic DNA and wildtype 60mer oligonucleotides. The oligonucleotides represent the wildtype sequence at the position of skip primer binding and enable detection of mispriming of the skip primers (Scheme I). The method was tested using published UBA52 and RPL23 primers known to misanneal at low frequency [1]. PCRs using those primers resulted in clear bands on gDNA. In contrast, in experiments using the skip primers used in our measurements, no products were found in PCR on gDNA or oligonucleotides, whereas PCRs on cDNA of fibroblasts resulted in the expected products (Figure 1). This implicates that misannealing with these primers may occur at even lower frequency than that published for the UBA52 and RPL23 primers (found to misanneal <1:1000).

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