SUMO-1 Modification Alters ADAR1 Editing Activity

Abstract

We identify ADAR1, an RNA-editing enzyme with transient nucleolar localization, as a novel substrate for sumoylation. We show that ADAR1 colocalizes with SUMO-1 in a subnucleolar region that is distinct from the fibrillar center, the dense fibrillar component, and the granular component. Our results further show that human ADAR1 is modified by SUMO-1 on lysine residue 418. An arginine substitution of K418 abolishes SUMO-1 conjugation and although it does not interfere with ADAR1 proper localization, it stimulates the ability of the enzyme to edit RNA both in vivo and in vitro. Moreover, modification of wild-type recombinant ADAR1 by SUMO-1 reduces the editing activity of the enzyme in vitro. Taken together these data suggest a novel role for sumoylation in regulating RNA-editing activity. INTRODUCTIONA defining feature of eukaryotic cells is the generation of protein diversity either posttranscriptionally by alternative splicing and RNA editing or posttranslationally by modification of amino acids in proteins. One of the most recently discovered posttranslational modification mechanism in eukaryotes involves the covalent attachment of the small ubiquitinlike modifier, SUMO, to target proteins. Modification of proteins by SUMO, or sumoylation, plays crucial regulatory roles in eukaryotes. Proteins known to be modified by SUMO include, among others, RanGAP1, PCNA, I B␣, p53, c-jun, topoisomerases, promyelocytic leukemia protein (PML), Sp100, and the mitogen-activated protein kinase kinase 1 (MEKK1). Many SUMO substrates are transcription factors and cofactors, or proteins implicated in DNA repair and replication (reviewed by Hay, 2001;Melchior et al., 2003;Seeler and Dejean, 2003;Hay, 2005). Although it is well established that SUMO can affect target protein function by altering its subcellular localization, activity, or stability, for many substrates the biological functions of sumoylation remain unknown.Sumoylation is a reversible and highly dynamic process that involves formation of an isopeptide bond between the C-terminus of SUMO and the ⑀-amino group of a lysine residue of the target protein. The most intensely studied human form of SUMO is the SUMO-1 protein, which is 48% identical to yeast Smt3 (Bayer et al., 1998;Mossessova and Lima, 2000). In vertebrates there are at least three additional proteins. SUMO-2 and SUMO-3 are ϳ45% identical to SUMO-1 (Saitoh and Hinchey, 2000), and SUMO-4 shows an 86% amino acid homology to SUMO-2 (Bohren et al., 2004). SUMO is conjugated to protein substrates via an ATP-dependent enzymatic pathway that is mechanistically similar to ubiquitination. The reaction requires a SUMO protease that removes four amino acids from the C-terminus of the 101-amino acid SUMO-1 precursor to generate the mature form; an heterodimeric SUMO-activating enzyme, SAE1/2; Ubc9, a SUMO-conjugating enzyme that ligates directly to its protein target; and an E3-like SUMO ligase (reviewed Melchior et al., 2003). Three SUMO E3s have been identified so far: the mammalian protein inhibitors of activated S...

Regulating the regulators: Serine/arginine‐rich proteins under scrutiny

“…Likewise, proteomic approaches revealed that RNA-binding proteins are the predominant group among small Ubiquitin-like modifier (SUMO) conjugation substrates, including several hnRNPs, SR family members and spliceosome components (50,51). Furthermore, SUMO conjugation has been found to regulate different aspects of mRNA metabolism such as pre-mRNA 3 0 end processing and RNA editing, by modifying the function of poly(A) polymerase, symplekin and CPSF-73 in the former case and ADAR1 in the latter (52,53). It would be interesting to elucidate whether Ub or Ubl conjugation, in particular SUMO, could affect SR protein activities.…”

The serine/arginine-rich protein SF2/ASF regulates protein sumoylation

“…An effect of sumoylation on the activity of different RNAbinding proteins has been reported (57,60,61), and sumoylation has been shown to regulate mRNA 3′-end processing (61,62). SUMO modification of nucleolar proteins controls their subnuclear distribution and members of the SENP family are concentrated within the nucleolus.…”

Dimerization of ADAR2 is mediated by the double-stranded RNA binding domain

“…To confirm that the signals obtained were not simply due to random associations between overexpressed proteins located in a small compartment, we used a nucleolar protein, nucleolin, which is not expected to interact with the ADARs. Electron and fluorescence microscopy have suggested that nucleolin localizes in the dense fibrillar and granular components of the nucleoli (Biggiogera et al 1990), while ADAR2 localizes to the periphery of the dense fibrillar component (Desterro et al 2003(Desterro et al , 2005. With quantitative confocal microscopy, we observe at least significant colocalization of ADAR2 and nucleolin within the nucleoli (data not shown), and when BRET 2 signals were measured between Rluc-ADAR2 and GFP 2 fused to nucleolin, the signal also increased with increased GFP 2 : Rluc ratio (Fig.…”