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.
Highlights
Cotranscriptional splicing of human pre-mRNA involves the interaction of several distinct protein factors and 5 snRNAs with sequences specific to the pre-mRNA [1]
Splicing noise frequencies were measured by real-time quantitative PCR (RT-qPCR) of the regular NF1, PCGF2, RABAC1 and AATF transcripts in relation to the products without the skipped exons (NF1-'38, NF1-'39, NF1-'46, NF1-'52, PCGF2-'10, RABAC1-'4 and AATF-'3)
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)
Summary
Cotranscriptional splicing of human pre-mRNA involves the interaction of several distinct protein factors and 5 snRNAs with sequences specific to the pre-mRNA [1]. Errors resulting for example in transcripts lacking one or more cassette exons rarely occur in splicing [3] Such erroneous transcripts were first observed in low temperature (cold shock) treated peripheral blood cells whilst screening for NF1 mutations at RNA level [4,5]. Multi-exon skipping was found at very low frequencies in NF1, TSC2, RPL23 and UBA52 in cultured human cells [1,6,7] It is debated whether these erroneous transcripts (splicing noise) are caused by a stochastic missing exon recognition, insufficient fidelity of transcription, inaccuracy of the splicing machinery or somatic mutations in single cells in splice regulating sequences or genes such as SC2 [1,6,7,8].
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
