Specific phosphorylation of SR proteins by mammalian DNA topoisomerase I

Abstract

Several metazoan splicing factors are characterized by ribonucleoprotein (RNP) consensus sequences and arginine-serine repeats (RS domain) which are essential for their function in splicing. These include members of the SR-protein family (SC35, SF2/ASF), the U1 small nuclear (sn) RNP protein (U1-70K) and the U2 snRNP auxiliary factor (U2AF). SR proteins are phosphorylated in vivo and the phosphorylation state of U1-70K's RS domain influences its splicing activity. Here we report the purification of a protein kinase that is specific for SR proteins and show that it is DNA topoisomerase I. This enzyme lacks a canonical ATP-binding motif but binds ATP with a dissociation constant of 50 nM. Camptothecin and derivatives, known to be specific inhibitors of DNA topoisomerase I, strongly inhibit the kinase activity in the presence of DNA and affect the phosphorylation state of SR proteins. Thus, DNA topoisomerase I may well be one of the SR protein kinases operating in vivo.

Nuclear Relocalization of the Pre-mRNA Splicing Factor PSF during Apoptosis Involves Hyperphosphorylation, Masking of Antigenic Epitopes, and Changes in Protein Interactions

“…A number of mammalian kinases have been shown to cause disassembly of nuclear speckles and relocalization of splicing factors, and to subsequently affect splicing factor activity. SRPK-1/2, Clk-1/2/3/4, cdc2-kinase, cyclin Ecdk2, topoisomerase I, U1 70-kDa associated kinase, cGMPdependent kinase, and lamin B-receptor kinase were all shown to phosphorylate SR proteins and other splicing factors in vitro (Woppmann et al, 1993;Gui et al, 1994;Colwill et al, 1996;Nikolakaki et al, 1996;Rossi et al, 1996;Nayler et al, 1997;Duncan et al, 1998;Kuroyanagi et al, 1998;Okamoto et al, 1998;Seghezzi et al, 1998;Wang et al, 1998;Koizumi et al, 1999;Wang et al, 1999). In addition, some of these kinases were shown to affect the splicing activity of such factors (Mermoud et al, 1994;Cao et al, 1997;Xiao and Manley, 1998;Prasad et al, 1999).…”

“…An alternative interpretation of the differential phosphorylation of PSF, during mitosis and particularly in apoptosis, would be that this protein has functions that are unrelated to splicing. Interestingly, topoisomerase I was shown to specifically phosphorylate SR proteins (Rossi et al, 1996). It therefore may be speculated that the removal of PSF from speckles via hyperphosphorylation is a means for its activation in other cellular functions.…”

Ready for a comeback of natural products in oncology

“…These protein interfaces may be targeted, if suitable assays can be developed. Topoisomerase I also carries a kinase activity important for the regulation of RNA splicing [69]. Although the link between the topoI kinase activity and cancer is still poorly understood, it is nevertheless clear that this is a potential new area for pharmacological intervention.…”

The topoisomerase 1-interacting protein BTBD1 is essential for muscle cell differentiation

“…4 Finally, Topo1 contributes to RNA splicing. 8,9 Regarding the Topo1 subcellular localization, a nuclear localization signal is present in its N-terminal region 10 and a direct interaction with nucleolin, a major nucleolar protein, has been shown by Bharti et al 11 thus indicating a probable nuclear localization. It is therefore not surprising that repression or overexpression of the Topo1 gene can lead to cell death, [12][13][14] indicating that preservation of a normal level of Topo1 is crucial for cell survival.…”