Arginine Methylation Controls the Subcellular Localization and Functions of the Oncoprotein Splicing Factor SF2/ASF

Abstract

Alternative splicing and posttranslational modifications (PTMs) are major sources of protein diversity in eukaryotic proteomes. The SR protein SF2/ASF is an oncoprotein that functions in pre-mRNA splicing, with additional roles in other posttranscriptional and translational events. Functional studies of SR protein PTMs have focused exclusively on the reversible phosphorylation of Ser residues in the C-terminal RS domain. We confirmed that human SF2/ASF is methylated at residues R93, R97, and R109, which were identified in a global proteomic analysis of Arg methylation, and further investigated whether these methylated residues regulate the properties of SF2/ASF. We show that the three arginines additively control the subcellular localization of SF2/ASF and that both the positive charge and the methylation state are important. Mutations that block methylation and remove the positive charge result in the cytoplasmic accumulation of SF2/ASF. The consequent decrease in nuclear SF2/ASF levels prevents it from modulating the alternative splicing of target genes, results in higher translation stimulation, and abrogates the enhancement of nonsense-mediated mRNA decay. This study addresses the mechanisms by which Arg methylation and the associated positive charge regulate the activities of SF2/ASF and emphasizes the significance of localization control for an oncoprotein with multiple functions in different cellular compartments.Pre-mRNA splicing is a required step for the expression of most human genes. Alternative splicing plays a key role in regulating gene expression and is a major source of protein isoform diversity. SR proteins are a family of closely related and highly expressed eukaryotic RNA-binding proteins that regulate both general and alternative splicing (22) and have additional roles in other aspects of gene expression (41). SF2/ ASF, a prototypical member of the SR protein family, has a modular structure with two RNA recognition motifs (RRMs), which recognize a 7-nucleotide exonic splicing enhancer (ESE) motif on pre-mRNA, and an RS domain with numerous repeats of serine-arginine dipeptides (Fig. 1A). SF2/ASF is required for general splicing, though sometimes interchangeably with other SR proteins (33), and also plays a role in the regulation of alternative splicing in a concentration-dependent manner, in part through the recognition of ESEs (16). In addition to its role as a broad-specificity splicing regulator, SF2/ ASF strongly enhances nonsense-mediated mRNA decay (NMD) upon overexpression (65), stimulates translation both in vivo and in vitro in an enhancer-dependent manner (50), and functions as an adaptor protein for mRNA export (26). Some of these additional functions of SF2/ASF reflect its ability to shuttle between the nucleus and the cytoplasm (13). Reflecting its multifaceted role as a regulator in various cellular processes, SF2/ASF is a potent oncoprotein whose overexpression by as little as 2-fold is sufficient to transform immortal rodent fibroblasts (30). Conversely, the knockdown of SF2/...

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

“…Not so long ago, a global analysis of methylation sites by heavy methyl SILAC (stable isotope labeling by amino acids in cell culture) has revealed that many different hnRNPs and also the SR protein SRSF1 and other splicing factors such as Tra2b, are methylation substrates (21). Human SRSF1 contains three methylated Arg residues (R93, R97 and R109) in the linker between RRM1 and RRM2, and the functional relevance of these methylations has been recently investigated by the Krainer laboratory (22). By mutating these three Arg residues within SRSF1 to Ala, they found that the triple-Ala mutant was still able to shuttle between the nucleus and the cytoplasm.…”

Alternative splicing factor or splicing factor‐2 plays a key role in intron retention of the endoglin gene during endothelial senescence

“…5A,B). Nevertheless, it is well known that some splicing factors, including ASF ⁄ SF2, are proteins that continuously shuttle between the cytoplasm and the nucleus depending on their serine phosphorylation or arginine methylation state (Sanford et al, 2005;Sinha et al, 2010). Therefore, we decided to analyze the subcellular distribution of ASF ⁄ SF2 by comparing young versus senescent HUVECs using confocal microscopy.…”

“…To assess the role of ASF ⁄ SF2 in the cytoplasmic alternative splicing, phosphorylation-and methylation-deficient versions of this factor that target the cytosol were used (Cazalla et al, 2002;Sinha et al, 2010). Thus, ASF ⁄ SF2-KS and ASF ⁄ SF2-A 1 A 2 A 3 cytoplasmic mutants were co-transfected with the MinigEND construction in HEK293T cells (Fig.…”

“…This different localization depends on the phosphorylation state of the RS domain in serine residues, which influences the functions of ASF ⁄ SF2 (Sanford et al, 2009). Furthermore, it has recently been reported that there are three arginine residues susceptible to methylation in the glycine-rich linker between the RMM motifs, which also condition the cytoplasmic accumulation of ASF ⁄ SF2 (Ong et al, 2004;Sinha et al, 2010). However, the physiological stimuli that regulate the activity and subcellular localization of ASF ⁄ SF2 remain largely unknown.…”

Alternative Splicing in Endothelial Senescence: Role of the TGF-β Co-Receptor Endoglin

“…ASF/SF2 is involved in both constitutive and alternative splicing processes. Although ASF/SF2 is mainly found in the nuclear speckles, it continuously shuttles between the nucleus and the cytoplasm depending on the phosphorylation and/or methylation states, which in turn determines its activity (Sanford et al, 2008;Sanford et al, 2005;Sinha et al, 2010). In this context, it has been recently reported the role of ASF/SF2 in the regulation of the S-endoglin intron retention during endothelial senescence (Blanco & Bernabeu, 2011).…”