The Conserved Intronic Cleavage and Polyadenylation Site of CstF-77 Gene Imparts Control of 3′ End Processing Activity through Feedback Autoregulation and by U1 snRNP

摘要

The human gene encoding the cleavage/polyadenylation (C/P) factor CstF-77 contains 21 exons. However, intron 3 (In3) accounts for nearly half of the gene region, and contains a C/P site (pA) with medium strength, leading to short mRNA isoforms with no apparent protein products. This intron contains a weak 5′ splice site (5′SS), opposite to the general trend for large introns in the human genome. Importantly, the intron size and strengths of 5′SS and pA are all highly conserved across vertebrates, and perturbation of these parameters drastically alters intronic C/P. We found that the usage of In3 pA is responsive to the expression level of CstF-77 as well as several other C/P factors, indicating it attenuates the expression of CstF-77 via a negative feedback mechanism. Significantly, intronic C/P of CstF-77 pre-mRNA correlates with global 3′UTR length across cells and tissues. In addition, inhibition of U1 snRNP also leads to regulation of the usage of In3 pA, suggesting that the C/P activity in the cell can be cross-regulated by splicing, leading to coordination between these two processes. Importantly, perturbation of CstF-77 expression leads to widespread alternative cleavage and polyadenylation (APA) and disturbance of cell proliferation and differentiation. Thus, the conserved intronic pA of the CstF-77 gene may function as a sensor for cellular C/P and splicing activities, controlling the homeostasis of CstF-77 and C/P activity and impacting cell proliferation and differentiation. Author Summary Autoregulation is commonly used in biological systems to control the homeostasis of certain activity, and cross-regulation coordinates multiple processes. We show that vertebrate genes encoding the cleavage/polyadenylation (C/P) factor CstF-77 contain a conserved intronic C/P site (pA) which regulates CstF-77 expression through a negative feedback loop. Since the usage of this intronic pA is also responsive to the expression of other C/P factors, the pA can function as a sensor for the cellular C/P activity. Because the CstF-77 level is important for the usage of a large number of pAs in the genome and is particularly critical for expression of genes involved in cell cycle, this autoregulatory mechanism has far-reaching implications for cell proliferation and differentiation. The human intron harboring the pA is large and has a weak 5′ splice site, both of which are also highly conserved in other vertebrates. Inhibition of U1 snRNP, which recognizes the 5′ splice site of intron, leads to upregulation of the intronic pA isoform of CstF-77 gene, suggesting that the C/P activity in the cell can be cross-regulated by splicing, leading to coordination between these two processes.