Abstract
The DNA damage response involves coordinated control of gene expression and DNA repair. Using deep sequencing, we found widespread changes of alternative cleavage and polyadenylation site usage on ultraviolet-treatment in mammalian cells. Alternative cleavage and polyadenylation regulation in the 3ʹ untranslated region is substantial, leading to both shortening and lengthening of 3ʹ untranslated regions of genes. Interestingly, a strong activation of intronic alternative cleavage and polyadenylation sites is detected, resulting in widespread expression of truncated transcripts. Intronic alternative cleavage and polyadenylation events are biased to the 5ʹ end of genes and affect gene groups with important functions in DNA damage response and cancer. Moreover, intronic alternative cleavage and polyadenylation site activation during DNA damage response correlates with a decrease in U1 snRNA levels, and is reversible by U1 snRNA overexpression. Importantly, U1 snRNA overexpression mitigates ultraviolet-induced apoptosis. Together, these data reveal a significant gene regulatory scheme in DNA damage response where U1 snRNA impacts gene expression via the U1-alternative cleavage and polyadenylation axis.
Highlights
Almost all eukaryotic mRNA precursors undergo a co-transcriptional modification at the 3ʹ end, which includes two coupled steps, cleavage and polyadenylation [1, 2]
Analysis of UV-induced APA by 3ʹ region extraction and deep sequencing (3ʹREADS) Previous studies indicated that 3ʹ end processing is regulated during DNA damage response (DDR) [25, 27,28,29,30,31,32,33]
To mitigate the effect of differential regulation of APA isoforms through mRNA decay in cytoplasm and in keeping with our previous work to study functional effect of DNA damage using nuclear RNA and factors [30,31,32,33,34], nuclear RNA was extracted and subjected to 3ʹREADS (Figure 1a), a recently developed deep sequencing method for analysis of APA isoform expression genome wide [17]
Summary
Almost all eukaryotic mRNA precursors undergo a co-transcriptional modification at the 3ʹ end, which includes two coupled steps, cleavage and polyadenylation [1, 2]. Well over half of the mammalian genes contain more than one pA, leading to expression of alternative cleavage and polyadenylation (APA) isoforms [11]. As 3ʹUTRs contain various cis elements for post-transcriptional control, such as microRNA target sites and AU-rich elements, 3ʹUTR-APA can significantly impact mRNA metabolism. Intron-APA can result in change of coding sequences of mRNA, impacting the proteome. The core mammalian C/P machinery and additional cis elements around the pA are responsible for the selection among APA sites [18,19,20]. In keeping with U1’s role in C/P, recent studies have shown that inhibition of U1 function leads to activation of intron-APA events, resulting in shorter transcripts [8, 21]
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