Characterization of posttranscriptional regulatory interactions in response to genotoxic stress
Cell Biology
Final Report Abstract
The DNA damage response (DDR) is a collective term for signal transduction pathways that sense, signal, and repair different types of DNA lesions in eukaryotic cells. Defects in these processes result in genome instability and may lead to neurological and immunological disorders, premature aging, and cancer progression. Immediate-to-early DDR relies on rapid post-translational modifications of DDR factors such as protein kinases, distinct components of repair machinery, and cell cycle regulators. On the cellular level, DDR impacts multiple layers of cell fate decisions from activation of specific DNA repair mechanisms, cell cycle progression/arrest, to apoptotic or senescent phenotypes. Importantly, a significant fraction of these decisions are mediated by a transcriptional response, which is largely under the control of TP53, a transcription factor that regulates expression of cell cycle regulators such as cyclin-dependent inhibitor protein 1A (CDKN1A/p21), apoptotic proteins (e.g., BAX, BBC3/PUMA), and DNA repair components. 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 protein-protein 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.
Publications
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(2017). DDX54 regulates transcriptome dynamics during DNA damage response. Genome Research 27, 1-16
Milek M, Imami K, Mukherjee N, De Bortoli F, Zinnall U, Hazapis O, Trahan C, Oeffinger M, Heyd F, Ohler U, Selbach M, Landthaler M
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(2018) Systematic Detection of Poly(A)+ RNA-Interacting Proteins and Their Differential Binding. Methods Mol Biol. 1649, 405-417
Milek M, Landthaler M.
