Poly(ADP-ribose) Binds to the Splicing Factor ASF/SF2 and Regulates Its Phosphorylation by DNA Topoisomerase I

Abstract

Human DNA topoisomerase I plays a dual role in transcription, by controlling DNA supercoiling and by acting as a specific kinase for the SR-protein family of splicing factors. The two activities are mutually exclusive, but the identity of the molecular switch is unknown. Here we identify poly(ADP-ribose) as a physiological regulator of the two topoisomerase I functions. We found that, in the presence of both DNA and the alternative splicing factor/splicing factor 2 (ASF/SF2, a prototypical SRprotein), poly(ADP-ribose) affected topoisomerase I substrate selection and gradually shifted enzyme activity from protein phosphorylation to DNA cleavage. A likely mechanistic explanation was offered by the discovery that poly(ADP-ribose) forms a high affinity complex with ASF/SF2 thereby leaving topoisomerase I available for directing its action onto DNA. We identified two functionally important domains, RRM1 and RS, as specific poly(ADP-ribose) binding targets. Two independent lines of evidence emphasize the potential biological relevance of our findings: (i) in HeLa nuclear extracts, ASF/SF2, but not histone, phosphorylation was inhibited by poly(ADP-ribose); (ii) an in silico study based on gene expression profiling data revealed an increased incidence of alternative splicing within a subset of inflammatory response genes that are dysregulated in cells lacking a functional poly(ADP-ribose) polymerase-1. We propose that poly(ADP-ribose) targeting of topoisomerase I and ASF/SF2 functions may participate in the regulation of gene expression. DNA topoisomerase I (topo I)2 is a constitutively expressed multifunctional enzyme that localizes at active transcription sites (1, 2). Its best known function is to control the topological state of DNA by relieving torsional stress that is generated following DNA strand separation during transcription, replication, and repair (3-5). The catalytic mechanism involves the formation of a DNA⅐topo I complex (cleavage complex) with the enzyme being covalently bound to the 3Ј-end of the cleaved DNA strand through a tyrosine-phosphate ester bond. Cleavage complexes are usually short-lived; their stabilization by compounds of the camptothecin (CPT) family of anticancer drugs may cause DNA strand break accumulation and eventually lead to cell death. Human topo I can relax both negative and positive supercoils by controlled rotation of the DNA strand downstream of the cleavage site followed by break resealing and restoration of an intact DNA duplex.In addition to relaxing supercoiled DNA, human topo I also plays a major role in pre-mRNA splicing, being endowed with a protein kinase activity targeted at a group of splicing factors of the serine-arginine (SR)-rich protein family (6). SR-proteins function both as components of the basal RNA splicing machinery and as regulators of alternative splicing (7, 8). Moreover, SR-proteins are involved in the control of mRNA transport and stability (8, 9) and contribute to the maintenance of genomic stability (10). SR-proteins are structurally characterized ...

Quantitative proteomics and dynamic imaging reveal that G3BP-mediated stress granule assembly is poly(ADP-ribose)-dependent following exposure to MNNG-induced DNA alkylation

“…Mapping of G3BP pADPr-binding indicates that the C-terminal glycine-and arginine-rich (GAR) region is a non-covalent pADPr-binding domain. The identification of a similar pADPr-binding GAR in the mRNA-binding protein ASF/SF2 supports our identification of the GAR region of G3BP as the pADPr-binding region (Malanga et al, 2008). Several reports indicate that GAR coordinates mRNA maturation events and mediates the nucleocytoplasmic trafficking of numerous RNA-binding proteins (reviewed by Godin and Varani, 2007).…”

“…In fact, pADPr is already known to interfere with domain essential to protein-protein interaction and has the ability to act as a scaffold molecule. For example, pADPr will most likely modulate activities of proteins within SGs, as seen with the ability of pADPr to shift activity of topoisomerase I towards ASF/SF2 (Yung et al, 2004;Malanga et al, 2008) or the postulated ability of pADPr to modulate the activity of hnRNPs, thereby affecting mRNA splicing (Ji and Tulin, 2009). Moreover, Leung et al demonstrate that pADPr modulates the assembly and maintenance of an mRNP-enriched structure.…”

Epigenomic reprogramming in inorganic arsenic-mediated gene expression patterns during carcinogenesis

“…Interestingly, PARP1 co-localizes with CTCF on chromatin; this complex, with CTCF-dependent automodification of PARP1, permits PARylation activity in the absence of DNA damage (137,139). Importantly, many splicing factors are regulated by PARylation (140)(141)(142)(143)(144) and any iAs-mediated inhibition of PARP1 binding to DNA not only affects the structural properties of chromatin but also the PARylation activities, which indirectly affect splicing decisions. In addition, the binding of proteins to DNA can be altered by the presence of iAs due to the high binding affinity of this metalloid for cysteine residues that are found in C4 and C3H1 zinc finger motif-containing proteins such as PARP1 (145)(146)(147).…”

Eukaryotic Replication Barriers: How, Why and Where Forks Stall

“…), accumulates at genes that are highly expressed during S-phase such as histone genes. The Top1 deficiency might affect fork progression in two ways; through Top1's role in releasing super-coiling between two types of converging forks, and through Top1's role in regulation of mRNP assembly, presumably by binding and phosphorylating splicing factors of the SR family (Rossi, et al 1996;Soret, et al 2003;Malanga, et al 2008). It has long been known that in bacterial genomes highly-expressed genes are oriented such that transcription does not interfere with replication and it has been proposed that this might also be true for a large fraction of the human genome (Huvet, et al 2007).…”