Human Tra2 proteins jointly control a CHEK1 splicing switch among alternative and constitutive target exons

Abstract

Alternative splicing—the production of multiple messenger RNA isoforms from a single gene—is regulated in part by RNA binding proteins. While the RBPs transformer2 alpha (Tra2α) and Tra2β have both been implicated in the regulation of alternative splicing, their relative contributions to this process are not well understood. Here we find simultaneous—but not individual—depletion of Tra2α and Tra2β induces substantial shifts in splicing of endogenous Tra2β target exons, and that both constitutive and alternative target exons are under dual Tra2α–Tra2β control. Target exons are enriched in genes associated with chromosome biology including CHEK1, which encodes a key DNA damage response protein. Dual Tra2 protein depletion reduces expression of full-length CHK1 protein, results in the accumulation of the DNA damage marker γH2AX and decreased cell viability. We conclude Tra2 proteins jointly control constitutive and alternative splicing patterns via paralog compensation to control pathways essential to the maintenance of cell viability.

Transformer2 proteins protect breast cancer cells from accumulating replication stress by ensuring productive splicing of checkpoint kinase 1

“…Inclusion of CHK1 exon 3 is thus needed to produce an mRNA that can be translated into a functional CHK1 protein. Consistent with this, in addition to the splicing defect in the CHK1 mRNA when Tra2α and Tra2β proteins were depleted, much lower levels of full length CHK1 protein were also produced [27]. Joint depletion of Tra2a and Tra2b also induced a concomitant increase in γH2AX staining, which demonstrates that DNA damage was accumulating in the MDA-MB-231 cells that were jointly depleted for Tra2 proteins -exactly the way that DNA damage accumulates after direct CHK1 depletion [27].…”

“…Although these data suggest that CHK1 mRNA is not productively translated when exon 3 is skipped, a further shorter isoform of the CHK1 mRNA has been described [27]. In MDA-MB-231 breast cancer cells, higher levels of Tra2β efficiently repress Tra2α protein expression.…”

“…Splicing of the poison exon into the Tra2a mRNA would block Tra2α expression from these poisoned mRNAs in MDA-MB-231 cells. However, when Tra2β levels are depleted by siRNA in MDA-MB-231 cells, this reduces splicing of the Tra2a poison exon, thereby resulting in a substantial increase in functional Tra2a mRNA, and the expression of Tra2α protein [27]. This feedback loop means that, overall, Tra2 protein activity might be expected to remain stable even when Fig.…”

“…A number of individual target genes for human Tra2β were known, including BRCA1 [25], but until recently the binding sites or functional targets of Tra2β protein across the entire human transcriptome were unknown. To globally address the functions of Tra2β in breast cancer cells, iCLIP was recently used to identify Tra2β RNA target sites in the MDA-MB-231 cell line [26,27]. The iCLIP method uses ultraviolet irradiation to cross link Tra2β to its target RNAs within cells, followed by immunoprecipitation and next generation sequencing of the recovered targets.…”

“…2 Tra2β binds to poison exons in the Tra2b and Tra2a genes. Tra2β binding sites were identified by iCLIP [27], and mapped onto the human genome. This screenshot from the UCSC genome browser shows high densities of Tra2β binding sites specifically above the poison exons within these genes [28] expression levels of Tra2β fall, because such falling levels would induce an increase in Tra2α levels that could compensate (Fig.…”