Highlights d CLK kinases are thermo-sensors reactive to subtle bodytemperature changes d Body temperature globally controls splicing and gene expression through CLKs d CLK homologs are evolutionarily adapted to diverse body/ growth temperatures d Wide functionality of CLKs: from TSD in reptiles to circadian biology in mammals

The cellular response to genotoxic stress is mediated by a well-characterized network of DNA surveillance pathways. The contribution of post-transcriptional gene regulatory networks to the DNA damage response (DDR) has not been extensively studied. Here, we systematically identified RNA-binding proteins differentially interacting with polyadenylated transcripts upon exposure of human breast carcinoma cells to ionizing radiation (IR). Interestingly, more than 260 proteins, including many nucleolar proteins, showed increased binding to poly(A) + RNA in IR-exposed cells. The functional analysis of DDX54, a candidate genotoxic stress responsive RNA helicase, revealed that this protein is an immediate-to-early DDR regulator required for the splicing efficacy of its target IR-induced pre-mRNAs. Upon IR exposure, DDX54 acts by increased interaction with a well-defined class of pre-mRNAs that harbor introns with weak acceptor splice sites, as well as by proteinprotein contacts within components of U2 snRNP and spliceosomal B complex, resulting in lower intron retention and higher processing rates of its target transcripts. Because DDX54 promotes survival after exposure to IR, its expression and/or mutation rate may impact DDR-related pathologies. Our work indicates the relevance of many uncharacterized RBPs potentially involved in the DDR.

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In the yeast U1 snRNP the Prp39/Prp42 heterodimer is essential for early steps of spliceosome assembly. In metazoans no Prp42 ortholog exists, raising the question how the heterodimer is functionally substituted. Here we present the crystal structure of murine PRPF39, which forms a homodimer. Structure-guided point mutations disrupt dimer formation and inhibit splicing, manifesting the homodimer as functional unit. PRPF39 expression is controlled by NMD-inducing alternative splicing in mice and human, suggesting a role in adapting splicing efficiency to cell type specific requirements. A phylogenetic analysis reveals coevolution of shortened U1 snRNA and the absence of Prp42, which correlates with overall splicing complexity in different fungi. While current models correlate the diversity of spliceosomal proteins with splicing complexity, our study highlights a contrary case. We find that organisms with higher splicing complexity have substituted the Prp39/Prp42 heterodimer with a PRPF39 homodimer.

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Human PRPF39 is a homolog of the yeast Prp39 and Prp42 paralogs. We have previously shown that human PRPF39 forms a homodimer that interacts with the CTD of U1C, mirroring the yeast Prp39/Prp42 heterodimer. We demonstrate here that PRPF39 knockdown in HEK293 cells affects many alternative splicing events primarily by reducing the usage of weak 5’ss. Additionally, PRPF39 preferentially binds to a GC-rich RNA, likely at the interface between its NTD and CTD. These data indicate that PRPF39 potentially recruits U1 snRNP to a weak 5’ ss, serving as a previously unrecognized alternative splicing factor. We further demonstrate that human TIA1 binds to U1C through its RRM1 and RRM3+Q domains but has no significant binding to PRPF39. Finally, all three human LUC7L isoforms directly interact with U1C. These results reveal significant parallels to the yeast U1 snRNP structure and support the use of yeast U1 snRNP as a model for understanding the mechanism of human alternative splicing.