Abstract
Mutation-driven activation of KRAS is crucial to cancer development. The human gene yields four mRNA splicing isoforms, 4A and 4B being translated to protein. Their different properties and oncogenic potential have been studied, but the mechanisms deciding the ratio 4A/4B are not known. To address this issue, the expression of the four KRAS isoforms was determined in 9 human colorectal cancer cell lines. HCT116 and SW48 were further selected because they present the highest difference in the ratio 4A/4B (twice as much in HCT116 than in SW48). Chromatin structure was analysed at the exon 4A, characteristic of isoform 4A, at its intronic borders and at the two flanking exons. The low nucleosome occupancy at exon 4A in both cell lines may result in a fast transcriptional rate, which would explain the general lower abundance of isoform 4A, also found in cells and tissues by other authors, but due to its similarity between both cell lines, chromatin structure does not influence alternative splicing. DNA methylation downstream exon 4A significantly differs in HCT116 and SW48 cells, but the CCCTC-binding factor, which affects the processivity of RNA polymerase and the alternative splicing, does not bind the differentially methylated sequences. Quantitative epigenetic analysis at mononucleosomal level revealed significant differences between both cell lines in H3K4me3, H3K27me3, H3K36me3, H3K9ac, H3K27ac and H4K20me1, and the inhibition of some histone-modifying enzymes alters the ratio 4A/4B. It can be concluded that the epigenetic modification of histones has an influence on the selection of isoforms 4A and 4B.
Highlights
Human KRAS locus is located in chromosome 12 (25,204,789-25,250,936) and is transcribed from the reverse strand
The low nucleosome occupancy at exon 4A in both cell lines may result in a fast transcriptional rate, which would explain the general lower abundance of isoform 4A, found in cells and tissues by other authors, but due to its similarity between both cell lines, chromatin structure does not influence alternative splicing
DNA methylation downstream exon 4A significantly differs in HCT116 and SW48 cells, but the CCCTC-binding factor, which affects the processivity of RNA polymerase and the alternative splicing, does not bind the differentially methylated sequences
Summary
Human KRAS locus is located in chromosome 12 (25,204,789-25,250,936) and is transcribed from the reverse strand. Four mRNA isoforms, which result from alternative splicing, are reported in the Ensembl Genome database (accession number ENSG00000133703). Two of these mRNA isoforms are translated to protein, giving rise to the well-known KRAS-4A and KRAS-4B products. The two remaining mRNA isoforms contain open reading frames and may be putatively translated, but the actual existence of their protein products has not been reported www.oncotarget.com to date. KRAS-4A and KRAS-4B are members of the Ras protein family, which includes the highly homologous HRAS and NRAS. All of these proteins display GTPase activity and are involved in signalling pathways that regulate many cellular processes, including cell proliferation
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