Dynamic Nucleocytoplasmic Shuttling of an Arabidopsis SR Splicing Factor: Role of the RNA-Binding Domains      

Abstract

Serine/arginine-rich (SR) proteins are essential nuclear-localized splicing factors. We have investigated the dynamic subcellular distribution of the Arabidopsis (Arabidopsis thaliana) RSZp22 protein, a homolog of the human 9G8 SR factor. Little is known about the determinants underlying the control of plant SR protein dynamics, and so far most studies relied on ectopic transient overexpression. Here, we provide a detailed analysis of the RSZp22 expression profile and describe its nucleocytoplasmic shuttling properties in specific cell types. Comparison of transient ectopic-and stable tissue-specific expression highlights the advantages of both approaches for nuclear protein dynamic studies. By site-directed mutagenesis of RSZp22 RNA-binding sequences, we show that functional RNA recognition motif RNP1 and zinc-knuckle are dispensable for the exclusive protein nuclear localization and speckle-like distribution. Fluorescence resonance energy transfer imaging also revealed that these motifs are implicated in RSZp22 molecular interactions. Furthermore, the RNA-binding motif mutants are defective for their export through the CRM1/XPO1/Exportin-1 receptor pathway but retain nucleocytoplasmic mobility. Moreover, our data suggest that CRM1 is a putative export receptor for mRNPs in plants.In eukaryotes, the protein-encoding genes are transcribed as long precursor molecules (pre-mRNAs) containing intervening sequences or introns. These introns are excised by a macromolecular complex, the spliceosome, consisting of five small nuclear ribonucleoproteins (U1, U2, U4/U6, and U5 snRNPs) and a large number of non-snRNP-associated proteins (Wahl et al., 2009). The Ser/Arg-rich (SR) proteins are described as a phylogenetically highly conserved family of non-snRNP essential splicing factors that mediate numerous events in pre-mRNA splicing (Reddy, 2007;Long and Caceres, 2009). In human, seven canonical SR proteins have been described with sizes ranging from 20 to 75 kD: SRp20, 9G8, ASF/SF2 (SRp30a), SC35 (SRp30b), SRp40, SRp55, and SRp75 (Bourgeois et al., 2004;Long and Caceres, 2009). SR proteins dynamically participate in spliceosome assembly through both protein-protein and protein-RNA interactions and have been recently reported to associate on nascent pre-mRNAs for cotranscriptional splicing (Das et al., 2007;Wahl et al., 2009). They promote the recruitment of the heterodimeric splicing factor U2AF and the U1 snRNP to the 3# and 5# splice sites, respectively. They also regulate alternative splicing by determining splice site selection, and this activity is antagonized mainly by heterogeneous nucleoproteins of the A and B groups (Graveley, 2000;Matlin et al., 2005). Although SR proteins appear to have redundant functions in vitro, individual proteins have specific activities in (pre-)mRNA metabolism (e.g. alternative splicing, mRNA export, translation) during the development of metazoans (Long and Caceres, 2009).SR proteins have a modular structure consisting of one or two N-terminal RNA recognition motifs (RRMs) and a C-ter...

Localization and Dynamics of Nuclear Speckles in Plants

“…AtRSZ22-GFP was shown to concentrate in the nucleolus upon phosphorylation inhibition, while other tested AtSRs did not (Tillemans et al, 2006). Later studies in root and pollen cells of stably transformed plants confirmed the accumulation of AtRSZ22-GFP within nucleoli upon phosphorylation inhibition (Rausin et al, 2010). In transiently expressing root cells, AtRS31-GFP, unlike other tested SRs, accumulated into concave cap-like structures surrounding regions devoid of fluorescence corresponding to the nucleoli following treatment with staurosporine.…”

“…A sensitive shuttling assay using FLIP and nuclear export inhibitors with cells transiently expressing FP fusions strongly suggested that RSZ22 is a nucleocytoplasmic shuttling SR splicing factor (Tillemans et al, 2006). The dynamics of RSZ22 was recently studied in stable transformants enabling tissue-specific expression of the transgene (Rausin et al, 2010). The results demonstrated that RSZ22 is a bona fide nucleocytoplasmic shuttling SR protein.…”

“…In further support that ATP depletion does not result from nonspecific general perturbation of the cellular environment, FRAP analysis with the deletion mutants of SR45 exhibited different localization and kinetic properties in response to ATP depletion (Ali and Reddy, 2006). In other studies, ATP depletion resulted in an accumulation of AtRSZ22-GFP within nucleoli, as had been demonstrated in phosphorylation inhibition, suggesting that the absence of RSZp22 mobility upon ATP depletion is linked to its phosphorylation state (Tillemans et al, 2006;Rausin et al, 2010). In animals, ATP depletion did not change the mobility of SR proteins (Phair and Misteli, 2000).…”

“…Other plant spliceosomal proteins (U1-70K, U2AF 35a , U2AF 35b ) are also localized to speckles in the nucleus (Wang and Brendel, 2006;Ali et al, 2008). Speckle components have been shown to shuttle continuously between speckles and nucleoplasm (Ali and Reddy, 2008b;Rausin et al, 2010). Their biogenesis is proposed to occur through selfassembly processes whereby a transient binding interaction to macromolecules will determine the size and shape of the speckles (Dundr and Misteli, 2010).…”

Alternative Splicing at the Intersection of Biological Timing, Development, and Stress Responses

“…Similarly, dehydration stress and heat stress increase the production of transcripts encoding the full-length SR45a protein, an atypical SR-like protein, relative to other splice variants (Gulledge et al, 2012). Furthermore, stress signals affect both the phosphorylation status and subcellular localization of Arabidopsis SR and splicingrelated proteins (Ali et al, 2003;Tillemans et al, 2005Tillemans et al, , 2006de la Fuente van Bentem et al, 2008;Koroleva et al, 2009;Rausin et al, 2010). Phosphorylation may affect the function of SF protein isoforms.…”