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.
Highlights
Alternative splicing—the production of multiple messenger RNA isoforms from a single gene—is regulated in part by RNA binding proteins
Western blot analysis confirmed this effect for two independent sets of small interfering RNA (siRNA) targeted against different parts of the respective messenger RNAs (mRNAs) (Supplementary Fig. 1)
TRA2B poison exon number of unique cDNAs mapping to each genomic region by the relative size of that region within the genome), we find that Tra2b binding is highly enriched within exons: 76.8% of Tra2b iCLIP tags mapped to exons (50UTR, 30UTR or ORF), while a further 20.4% mapped to non-coding RNAs (Supplementary Fig. 2c)
Summary
Alternative splicing—the production of multiple messenger RNA isoforms from a single gene—is regulated in part by RNA binding proteins. Endogenous Tra2b target RNAs have been identified using HITS-CLIP18, RIP-seq[28], shRNA depletion[29] and microarrays[17], but important fundamental questions still remain as to the identity of the biological targets and the functions of vertebrate Tra[2] proteins These include whether endogenous Tra2a and Tra2b proteins jointly control the same splicing targets, and if so what these shared targets are? Tra2a and Tra2b both activate splicing of the same model exons when overexpressed in transfected HEK-293 cells (suggesting redundant functions)[18], the Tra2a gene alone is not sufficient to maintain viability in Tra2b knockout mice (suggesting specific functions)[15] Another question relates to how Tra2a and Tra2b interact with each other? Poison exons introduce premature translation termination codons into mRNAs so as to inhibit translation of full-length proteins and are often regulatory[18,30,31,32], but whether Tra2a might reciprocally control Tra2b expression is not known
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
