Abstract
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.
Highlights
Pre-mRNA cleavage/polyadenylation (C/P) is a 39 end processing mechanism employed by almost all protein-coding genes in eukaryotes [1,2]
We show that vertebrate genes encoding the cleavage/polyadenylation (C/P) factor Cleavage stimulation Factor (CstF)-77 contain a conserved intronic C/P site which regulates CstF-77 expression through a negative feedback loop
Since the usage of this intronic pA is responsive to the expression of other C/P factors, the pA can function as a sensor for the cellular C/P activity
Summary
Pre-mRNA cleavage/polyadenylation (C/P) is a 39 end processing mechanism employed by almost all protein-coding genes in eukaryotes [1,2]. While the majority of alternative pAs are located in the 39-most exon, leading to regulation of 39 untranslated regions (39UTRs), about half of the genes have pAs located in introns [8], leading to changes in coding sequences (CDSs) and 39UTRs. Intronic pAs can be classified into two groups depending upon the splicing structure of the resultant terminal exon: composite terminal exon pA or skipped terminal exon pA. A composite terminal exon pA is located in a terminal exon which contains both exon and intron sequences. In this case, a 59 splice site (59SS) is located upstream of the pA. IgM mRNAs switch from using a 39most exon pA to an intronic pA during activation of B cells, which results in a shift in protein production from a membrane-bound form to a secreted form
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