Arginine methylation of the C-terminus RGG motif promotes TOP3B topoisomerase activity and stress granule localization

Abstract

DNA topoisomerase 3B (TOP3B) is unique among all mammalian topoisomerases for its dual activities that resolve both DNA and RNA topological entanglements to facilitate transcription and translation. However, the mechanism by which TOP3B activity is regulated is still elusive. Here, we have identified arginine methylation as an important post-translational modification (PTM) for TOP3B activity. Protein arginine methyltransferase (PRMT) 1, PRMT3 and PRMT6 all methylate TOP3B in vitro at its C-terminal arginine (R) and glycine (G)-rich motif. Site-directed mutagenesis analysis identified R833 and R835 as the major methylation sites. Using a methylation-specific antibody, we confirmed that TOP3B is methylated in cells and that mutation of R833 and R835 to lysine (K) significantly reduces TOP3B methylation. The methylation-deficient TOP3B (R833/835K) is less active in resolving negatively supercoiled DNA, which consequently lead to accumulation of co-transcriptionally formed R-loops in vitro and in cells. Additionally, the methylation-deficient TOP3B (R833/835K) shows reduced stress granule localization, indicating that methylation is critical for TOP3B function in translation regulation. Mechanistically, we found that R833/835 methylation is partially involved in the interaction of TOP3B with its auxiliary factor, the Tudor domain-containing protein 3 (TDRD3). Together, our findings provide the first evidence for the regulation of TOP3B activity by PTM.

The many lives of type IA topoisomerases

“…Although no systematic, side-by-side comparison of the activities of TOP3A and TOP3B have been performed, they do apparently display marked differences in their substrate preferences. In particular, TOP3B can fully relax a negatively supercoiled plasmid, which strikingly contrasts with the weak relaxation activities of all other TOP3s in general and of TOP3A in particular (171,(179)(180)(181). Although these observations are in contradiction with the initial biochemical characterization of Drosophila TOP3B that seemed to indicate inefficient DNA relaxation, they raise the interesting possibility that this isoform exhibits unique core DNA topoisomerase properties (182).…”

“…These phenotypes, also observed when TOP1 and TOP2B are defective, suggest that TOP3B has a role in releasing topological constraints associated with transcription (170). Such a role is supported by the efficient relaxation activity of TOP3B in vitro, which appears to be specifically targeted to sites of transcription in vivo where TOP3B acts to prevent the accumulation of R-loops (60,171).…”

“…Although one cannot exclude the possibility that TOP3B plays a direct role in DNA replication or repair, this increase in DNA damage may be directly related to the function of TOP3B in preventing R-loop accumulation during transcription. Indeed, Rloops have been shown to represent a threat to genome stability, in particular in proliferating cells where they can clash with the replication machinery (60,171,176).…”

Arginine methylation augments Sbp1 function in translation repression and decapping

“…Our current results are based on usage of antibody specific for mono-methyl arginine. Besides using the mono-methyl arginine antibody, we have also attempted using di-methylarginine antibodies such as ICP0810, Immunechem [30] and 13522, CST [31] but unlike 8711 (CST) we did not get consistent results both in vivo and in vitro for Sbp1 with these antibodies hence we are unable to comment on status of dimethylated arginines in Sbp1 in yeast cells. The cross-reactivity with the yeast total lysate (input) also has been very poor and inconsistent.…”

Topoisomerase 3B (TOP3B) DNA and RNA Cleavage Complexes and Pathway to Repair TOP3B-linked RNA and DNA Breaks

“…TOP3B localizes to both the nucleus and cytoplasm under steady-state condition (more abundant in the cytosol) as it facilitates both DNA and RNA metabolic processes inside cells (Stoll et al, 2013;Xu et al, 2013). Inside the nucleus, TOP3B in association with the scaffolding protein TDRD3 (Tudor Domain Containing 3) is recruited to promoter regions of target genes (NRAS, DDX5 and c-MYC) and has been proposed to facilitate transcription by relaxing hypernegatively supercoiled DNA and resolving R-loops (non-canonical nucleic acid structure consisting of RNA-DNA hybrids and displaced single-stranded DNA) (Goto-Ito et al, 2017;Huang et al, 2018a;Siaw et al, 2016;Yang et al, 2014;Zhang et al, 2019). TDRD3 also associates with the other well-known mRNA-binding protein FMRP (Fragile X Mental Retardation Protein) having roles in neuronal translation via its C-terminal FMRP Interacting Motif (FIM).…”