Dynamic Distribution and Interaction of the Arabidopsis SRSF1 Subfamily Splicing Factors

Ser/Arg-rich (SR) proteins are essential nucleus-localized splicing factors. Our prior studies showed that Arabidopsis (Arabidopsis thaliana) RSZ22, a homolog of the human SRSF7 SR factor, exits the nucleus through two pathways, either dependent or independent on the XPO1 receptor. Here, we examined the expression profiles and shuttling dynamics of the Arabidopsis SRSF1 subfamily (SR30, SR34, SR34a, and SR34b) under control of their endogenous promoter in Arabidopsis and in transient expression assay. Due to its rapid nucleocytoplasmic shuttling and high expression level in transient assay, we analyzed the multiple determinants that regulate the localization and shuttling dynamics of SR34. By site-directed mutagenesis of SR34 RNA-binding sequences and Arg/Ser-rich (RS) domain, we further show that functional RRM1 or RRM2 are dispensable for the exclusive protein nuclear localization and speckle-like distribution. However, mutations of both RRMs induced aggregation of the protein whereas mutation in the RS domain decreased the stability of the protein and suppressed its nuclear accumulation. Furthermore, the RNA-binding motif mutants are defective for their export through the XPO1 (CRM1/Exportin-1) receptor pathway, but retain nucleocytoplasmic mobility. We performed a yeast two hybrid screen with SR34 as bait and discovered SR45 as a new interactor. SR45 is an unusual SR splicing factor bearing two RS domains. These interactions were confirmed in planta by FLIM-FRET and BiFC and the roles of SR34 domains in protein-protein interactions were further studied. Altogether, our report extends our understanding of shuttling dynamics of Arabidopsis SR splicing factors.Ser/Arg-rich (SR) protein is the collective name given to a family of highly conserved splicing factors in Eukaryotes that regulate constitutive and alternative precursor mRNA splicing. SR proteins contain at least one RNA recognition motif (RRM) and an Arg/Ser-rich (RS) C-terminal domain (Manley and Krainer, 2010; Califice et al., 2012). The RRM appears to determine RNA-binding specificity, while the RS domain is involved in protein-protein and protein-RNA interactions (Shen et al., 2004). In human, twelve SR proteins have been described based on a set of formal criteria (Manley and Krainer, 2010). SR proteins have a modular organization: some SR proteins contain two RRMs while others contain a Zn-knuckle, which contributes to RNA binding. The activity of SR proteins is regulated by posttranslational modifications, such as Ser phosphorylation/dephosphorylation and Arg methylation. At steady-state, SR proteins accumulate in subnuclear speckles, which correspond to storage, assembly, and/or modification compartments for splicing factors. Several human SR proteins shuttle between the nucleus and the cytoplasm, and this dynamic shuttling is linked to their postsplicing activities in mRNA export, stability, and translation (Long and Caceres, 2009). The multiple roles and mechanisms of action of mammalian SR proteins have been extensively studied (for review, see Long and Caceres, 2009; Zhong et al., 2009; Kornblihtt et al., 2013; Änkö, 2014).The number of genes encoding SR proteins is higher in plants compared with metazoan. Plant genomes contain SR proteins homologous to the animal prototypes SRSF1/SRSF2/SRSF7, as well as plant-specific ones (Barta et al., 2010; Califice et al., 2012). Arabidopsis SR splicing factors localize into nuclear irregular dynamic domains similar to speckles, with no, only partial or complete colocalization (Tillemans et al., 2005; Lorković et al., 2008; Reddy et al., 2012). The functions of plant SR factors in postsplicing events remain unknown, though a nucleocytoplasmic shuttling activity has been described for RSZ22, a prototypic member of the SRSF7 subgroup (1 RRM, 1 Zn-knuckle) of Arabidopsis (Arabidopsis thaliana) SR protein family (Tillemans et al., 2006; Rausin et al., 2010).The nucleocytoplasmic transport of RNA and proteins occurs through nuclear pore complexes (NPCs), which require importin and exportin receptors (karyopherins or Kap) for trafficking of molecules larger than 40–90 kD. Kap often binds to cargo molecules that carry either nuclear localization signals (NLS) for nuclear import or nuclear export signals (NES) for nuclear export (Boruc et al., 2012). The best-known import pathway is mediated by the importin-α/β Kap that binds to NLS. Kap-β2 (or Transportin-SR, TRN-SR) was shown to function as the nuclear import receptor for human SRSF1 and SRSF2, and several Arabidopsis SR proteins (Yun et al., 2003; Xu et al., 2011). The human TRN-SR has recently been shown to embrace both the RRM and RS domains of SRSF1 for nuclear import (Maertens et al., 2014).XPO1 (Exportin-1, also named CRM1 in yeast Saccharomyces cerevisiae]) is a well-characterized mammalian nuclear export receptor which recognizes Leu-rich NES (φ-X_2-3-φ-X_2-3-φ-X-φ, where φ is L, V, I, F, or M and X is any amino acid) on proteins implicated in snRNA and rRNA export (Natalizio and Wente, 2013). XPO1/CRM1 was also shown to mediate the export of unspliced (or partially spliced) viral mRNAs and of a small subset of mRNAs. XPO1 recruitment to mRNA is mediated by single adaptor proteins including Leu-rich pentatricopeptide repeat proteins (LRPPRC) and HuR (Natalizio and Wente, 2013). Apart from this, the bulk of mRNA is exported by the nonkaryopherin heterodimer Nxf1-Nxt1 (TAP-p15) in metazoans (Mex67-Mtr2 in yeast). The shuttling SR proteins are known to promote messenger ribonucleoprotein (mRNP) export through NPCs when dephosphorylated by interacting with export factor Nxf1 (Huang et al., 2003). Several human SR proteins are also part of the exon junction complex (EJC) deposited upstream of exon-exon junctions after splicing, consistent with a role of SR proteins in mRNP export and nonsense mediated RNA decay (Singh et al., 2012). The RS domain is necessary but not sufficient for the cytoplasmic export of shuttling SR proteins (Cáceres et al., 1997).We previously identified RSZ22 as a shuttling splicing factor whose nuclear export is at least partly controlled by the XPO1-dependent export pathway (Tillemans et al., 2006; Rausin et al., 2010). Mutating conserved residues within the RNA-binding motifs of this specific SR protein highlighted the in vivo dependence of RNA binding for proper subcellular dynamics (Rausin et al., 2010). However, the role of the different protein domains in directing the cellular dynamics may vary among SR proteins, and the role of the RS domain of RSZ22 had not been investigated. It is also unknown whether XPO1-dependent nuclear export also includes other Arabidopsis SR proteins. A more global understanding of the molecular mechanisms underlying the nucleocytoplasmic transport of plant SR factors therefore required further investigation.Here, we functionally characterized the four Arabidopsis SR proteins of the SRSF1 subfamily (orthologs of mammalian SRSF1) that contain two conserved RRM domains (Califice et al., 2012). We studied the expression profiles of SR30, SR34, SR34a, and SR34b, and attempted to investigate their shuttling activity. Among these SR proteins, SR30 showed a less active nuclear export rate, and SR34b protein was not detectable in any expression assay. Because of its stability and rapid shuttling, we further focused on the SR34 protein by generating a series of mutant versions of the RRMs and RS domains. We established the overall requirement of these protein domains to retain nucleocytoplasmic shuttling activity. Yeast two-hybrid (Y2H) assays also revealed strong interactions between SRSF1 subfamily members (SR30, SR34, and SR34a) and SR45, an atypical SR protein (two RS domains). We also investigated the importance of SR34 domains in protein-protein interactions. Collectively, our findings provide a more detailed mechanistic understanding of the role of the structural determinants regulating SR proteins dynamics, and insights into protein domain function in in vivo interactions.