C-abl And Regulate Alternative Splicing And Nonsense Mutation-mediated Mrna Degradation

c-abl is the cellular homolog of the v-abl oncogene of the Abelson murine leukemia virus and is ubiquitously expressed in mammal cells. The c-abl coding protein c-Abl is a member of non-receptor tyrosine kinases and contains several domains, including(from N to C terminal) SH3, SH2, ATP binding region, tyrosine kinases domain(TK), proline-rich motifs, nuclear localization signal(NLS), DNA binding domain, G-Actin and F-Actin binding domain, nuclear export signal(NES).The nuclear c-Abl is activated upon DNA damage stress such as IR and cisplatin through the ATM and DNA-PK signaling pathway, and induces cell cycle arrest. During the stressed damages response to UV or TNF--α, c-Abl interacts with IκBαand induces apoptosis.The abelson related gene Arg is another member of abelson family, which is highly conserved in sequence with abelson.Previous studies have showed that c-Abl interacts with an alternative splicing factor, implies that c-Abl may involved in the alternative splicing regulation.Splicing is the process by which introns are removed from pre-mRNA and exons are ligated to form a mature mRNA in eukaryotes. Alterations in splice choice results in different mRNA variants, termed as alternative splicing. Alternative splicing is carried out by the spliceosome, a macromolecular complex that assembled from five snRNPs(U1,U2,U4,U5,U6) and numerous accessory proteins. The spliceosome uses multiple signals to recognize the splice sites precisely and catalyze the splicing reaction effectively.Alternative splicing plays an important role in gene regulation and protein diversity, and involves lots of cis-elements and trans- factors.Alternative splicing produces new proteins, and changes the binding properties, enzymatic activities, intracellular localization, signaling activities and stability of proteins. Errors in splicing can cause human diseases.Alternative splicing can change the mRNA open reading frame, and introduce premature termination codons(PTCs) into mRNA. mRNAs that contain PTCs can be degraded by nonsense-mediated decay(NMD), which is an important mechanism for the regulation of gene expression. The NMD surveillance system degrades the PTC containing mRNAs before they are translated into truncated proteins, protecting the cells from the potentially toxic effects of nonsense codons. We obtained a c-Abl knockdown mutant(KD) from human breast cancer cell line MCF-7 by RNA interference. Total RNA was purified from cultured cells, and Affymetrix Exon Array was used to study alternative splicing between mutant and wild cell lines. After several specific data processing steps, genes with important functions were selected for validating by RT-PCR. In our experiments, 8 genes in KD/MCF were validated, which confirmed the conjecture that c-Abl participates in alternative splicing. We then analyzed the genes'functions and discussed the relationship between the alternative splicing events and the function of c-Abl.Also, we found 3 novel splicing variants among those regulated genes, including IL4R, PSMB10 and SRRM1, while all the novel splicing isoforms generated premature termination codons(PTCs) by frame shifting. IL4R skipped exon 6, and PSMB10 and SRRM1/SRm160 had intron retentions in exon 6 - exon 7 and exon 3 - exon 4 respectively.The mRNAs harboured PTCs("PTC+mRNAs") of gene IL4R,PSMB10 and SRRM1 were up-regulated in the c-abl knockdown mutant cell line KD compared to wild type MCF-7. Further studying showed that PTC+ variants in SRRM1 and PSMB10 were up-regulated in murine MEF mutant cell line DKO(abl and arg double-knockout). The results suggest that knockdown or knockout of c-Abl would increase the "PTC+mRNAs" during genes'alternative splicing.RNAi and protein synthesis inhibition experiments demonstrated that the novel PTC-contained variant of gene SRRM1/SRm160 led the mRNA to degrade through NMD. Further experiment showed that the expression of this variant was cell-cycle specific, which highly expressed in G1 phase and lower in S and G2/M phases.We then analyzed 21 PTC-containing genes in murine DKO/MEF which had confirmed targeted to nonsense-mediated mRNA decay(NMD). The results demonstrated that the PTC-mRNAs in up to 10 genes are up-regulated in DKO, suggesting that c-Abl involved in the regulation of splicing of PTC-mRNAs and, probably NMD.