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

Maria Malanga,Alicja Czubaty,Agnieszka Girstun,Krzysztof Staron,Felix R Althaus

doi:10.1074/jbc.m709495200

Maria Malanga, Alicja Czubaty + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m709495200

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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 SR-protein), 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.

Highlights

Formation of a DNA1⁄7topo I complex with the enzyme being covalently bound to the 3Ј-end of the cleaved DNA strand through a tyrosine-phosphate ester bond
In addition to relaxing supercoiled DNA, human topoisomerase I (topo I) 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]
Protein targeting by PAR polymerases (PARPs)-bound polymers via non-covalent, yet specific interactions, is emerging as an important regulatory mechanism for diverse biological functions, including transcription, DNA damage signaling and checkpoint activation, proteasomal histone degradation, and mitotic spindle formation [22, 23]. topo I bears three PAR-binding sites localized in domains that are critical for the catalytic activity of the enzyme on DNA and for its regulation [24]

Summary

MATERIALS AND METHODS

Purified human topo I was obtained from TopoGen. This enzyme undergoes spontaneous conversion into a 70-kDa form lacking the N-terminal domain (⌬N-topo I). Reaction mixtures (15 ␮l) were assembled on ice and contained 50 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.01% Triton X-100, 20 ␮M CPT, 0.2 mg/ml bovine serum albumin, 2% glycerol, human topo I (0.2– 0.27 pmol). After incubation at 37 °C for 20 min, reaction was stopped by adding concentrated Laemmli buffer; ASF/SF2 phosphorylation was visualized by autoradiography, after electrophoretic separation on 10% polyacrylamide gels (SDSPAGE). Double DNA Cleavage/Protein Phosphorylation Assay—Recombinant human topo I (0.2 pmol) and recombinant His-ASF/ SF-2 (2.8 pmol) were incubated at 37 °C for 20 min, in 50 mM Tris-HCl, pH 8.0, containing 0.1 mM EDTA, 1 mM DTT, 10 mM MgCl2, 0.01% Triton X-100, 20 ␮M CPT, 0.2 mg/ml bovine serum albumin, 2% glycerol, 32P-5Ј-end-labeled ds-oligonucleotide (0.034 pmol, 0.5–1.1 ϫ 106 dpm/pmol), ␥-[32P]ATP (1.5 pmol, 0.3 ϫ 106 dpm/pmol). Genes coding for at least one expressed alternatively spliced isoform were taken as positive and expressed as the percentage of total analyzed genes

RESULTS

DISCUSSION

Mouse liver

Mouse heart