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.
Highlights
Serine/arginine-rich (SR) proteins are essential nuclear-localized splicing factors
We previously developed a fluorescence loss in photobleaching (FLIP)-shuttling assay to investigate the dynamics of SR proteins in vivo after transient expression
The human nucleocytoplasmic shuttling SR proteins function as regulators of mRNA export, stability, and translation (Zhong et al, 2009)
Summary
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. 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). Several nucleocytoplasmic trafficking pathways have been described that require so-called karyopherins for trafficking of molecules larger than approximately 40 to 90 kD These specific transport receptors bind to cargo molecules that carry either nuclear localization signals for nuclear import or nuclear export signals (NES) for nuclear export (Wang and Brattain, 2007; Xu and Meier, 2008). The human 9G8-shuttling SR protein associates with mRNA, providing an adapter function in recruiting the export receptor Nxf (Huang et al, 2003)
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