SUMOylation Regulates TDP-43 Splicing Activity and Nucleocytoplasmic Distribution

Abstract

The nuclear RNA-binding protein TDP-43 forms abnormal cytoplasmic aggregates in the brains of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients and several molecular mechanisms promoting TDP-43 cytoplasmic mislocalization and aggregation have been proposed, including defects in nucleocytoplasmic transport, stress granules (SG) disassembly and post-translational modifications (PTM). SUMOylation is a PTM which regulates a variety of cellular processes and, similarly to ubiquitination, targets lysine residues. To investigate the possible regulatory effects of SUMOylation on TDP-43 activity and trafficking, we first assessed that TDP-43 is SUMO-conjugated in the nuclear compartment both covalently and non-covalently in the RRM1 domain at the predicted lysine 136 and SUMO-interacting motif (SIM, 106–110 residues), respectively. By using the SUMO-mutant TDP-43 K136R protein, we demonstrated that SUMOylation modifies TDP-43 splicing activity, specifically exon skipping, and influences its sub-cellular localization and recruitment to SG after oxidative stress. When promoting deSUMOylation by SENP1 enzyme over-expression or by treatment with the cell-permeable SENP1 peptide TS-1, the cytoplasmic localization of TDP-43 increased, depending on its SUMOylation. Moreover, deSUMOylation by TS-1 peptide favoured the formation of small cytoplasmic aggregates of the C-terminal TDP-43 fragment p35, still containing the SUMO lysine target 136, but had no effect on the already formed p25 aggregates. Our data suggest that TDP-43 can be post-translationally modified by SUMOylation which may regulate its splicing function and trafficking, indicating a novel and druggable mechanism to explore as its dysregulation may lead to TDP-43 pathological aggregation in ALS and FTD.

Proteomics Elucidating Physiological and Pathological Functions of TDP-43

“…Although site-directed mutagenesis is a powerful tool to stimulate small post-translational modifications of amino acids such as serine/threonine phosphorylation and lysine acetylation, respectively, the aspartate/glutamate and glutamine substitutions are not identical to the phosphorylated or acetylated residues. Although molecular dynamics simulations suggested little impact of the K136R substitution on the local structure within the RNA-binding domain [166], subtle clashes with bound RNA cannot be ruled out [125]. Nevertheless, amber suppression confirmed the results of site-directed mutagenesis, showing that K136 acetylation reduced RNA binding and splicing activity.…”

“…As for de-ubiquitinating enzymes, only the rather general UBPY/USP8 has been described so far, counteracting ubiquitinylated TDP-43 pathology in cell and fly models [44]. TDP-43 is also a target for the small ubiquitin-related modifier (SUMO ) [165,166]. A SUMOylation consensus site comprises K136 within the RNA binding domain.…”

“…Inhibition of SUMOylation with anacardic acid treatment of K136R substitution reduced TDP-43 aggregation and cytotoxicity [165]. Mutant [K136R]TDP-43 showed reduced binding and splicing activity towards several target mRNAs [166]. However, it should be noted that K136 is also subject to lysine acetylation (see below), so it has to be carefully determined if SUMOylation or acetylation account for effects observed by K136 mutagenesis.…”

SUMOylation regulates pre‐mRNA splicing to overcome DNA damage in fungi

“…However, our study showed that the SUMOylation of FgSR controls DDR gene transcription not only by modulating its occupancy on target gene promoter resulting from nuclear‐cytoplasmic partitioning but also by regulating pre‐mRNA splicing, which provides a novel insight into the functions of SUMOylation of transcription factors. Previous studies have reported that SUMOylation is involved in pre‐mRNA splicing mainly via modifying splicing‐related proteins (Liu et al ., 2015; Pozzi et al ., 2017, 2018; Maraschi et al ., 2021). To our knowledge, this is the first report of SUMO modification of transcription factors participates in pre‐mRNA splicing regulation.…”