Abstract
Global shortening of 3′UTRs by alternative polyadenylation (APA) has been observed in cancer cells. However, the role of APA in cancer remains unknown. CCND1 is a proto-oncogene that regulates progression through the G1-S phase of the cell cycle; moreover, it has been observed to be switching to proximal APA sites in cancer cells. To investigate the biological function of the APA of CCND1, we edited the weak poly(A) signal (PAS) of the proximal APA site to a canonical PAS using the CRISPR/Cas9 method, which can force the cells to use a proximal APA site. Cell cycle profiling and proliferation assays revealed that the proximal APA sites of CCND1 accelerated the cell cycle and promoted cell proliferation, but UTR-APA and CR-APA act via different molecular mechanisms. These results indicate that PAS editing with CRISPR/Cas9 provides a good method by which to study the biological function of APA.
Highlights
Most human genes contain more than one poly (A) site, which leads to the prevalence of alternative polyadenylation (APA)[1]
To investigate the effects of APA on endogenously expressed CCND1, we performed poly(A) signal (PAS) editing with CRISPR/Cas[9] in the 293T cell line to express truncated CCND1a and CCND1b
Biological function of APA of CCND1 through PAS editing with the CRISPR/Cas[9] system, a method that can be used for future studies of APA function
Summary
Most human genes contain more than one poly (A) site, which leads to the prevalence of alternative polyadenylation (APA)[1]. In tumor cell lines and cancer patients, two major isoforms of CCND1 have been identified: CCND1a, which contains exons 1–5, and CCND1b, which ends with a longer exon 4 and is created by CR-APA using poly(A) sites within intron 420–23. Two mantel cell lymphoma patients harbor mutations in exon 5 (position 304 bp downstream of the stop codon), that produce a novel poly(A) signal (PAS: AAUAAA) and an isoform of CCND1a mRNA with a shorter 3′UTR (truncated CCND1a)[20]. Using the 3′ end sequencing technologies SAPAS and IVT-SAPAS, we observed expression of truncated CCND1a, albeit without a PAS, at this APA site in the breast cancer cell lines MCF7 and MB231 and in the mammary epithelial cell line MCF10A24,25. To investigate the effects of APA on endogenously expressed CCND1, we performed PAS editing with CRISPR/Cas[9] in the 293T cell line to express truncated CCND1a and CCND1b. Biological function of APA of CCND1 through PAS editing with the CRISPR/Cas[9] system, a method that can be used for future studies of APA function
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