Dynamic nucleocytoplasmic shuttling of an Arabidopsis SR splicing factor: role of the RNA-binding domains.

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.

Insights into nuclear organization in plants as revealed by the dynamic distribution of Arabidopsis SR splicing factors.

Serine/arginine-rich (SR) proteins are splicing regulators that share a modular structure consisting of one or two N-terminal RNA recognition motif domains and a C-terminal RS-rich domain. We investigated the dynamic localization of the Arabidopsis thaliana SR protein RSZp22, which, as we showed previously, distributes in predominant speckle-like structures and in the nucleolus. To determine the role of RSZp22 diverse domains in its nucleolar distribution, we investigated the subnuclear localization of domain-deleted mutant proteins. Our results suggest that the nucleolar localization of RSZp22 does not depend on a single targeting signal but likely involves different domains/motifs. Photobleaching experiments demonstrated the unrestricted dynamics of RSZp22 between nuclear compartments. Selective inhibitor experiments of ongoing cellular phosphorylation influenced the rates of exchange of RSZp22 between the different nuclear territories, indicating that SR protein mobility is dependent on the phosphorylation state of the cell. Furthermore, based on a leptomycin B- and fluorescence loss in photobleaching-based sensitive assay, we suggest that RSZp22 is a nucleocytoplasmic shuttling protein. Finally, with electron microscopy, we confirmed that RSp31, a plant-specific SR protein, is dynamically distributed in nucleolar cap-like structures upon phosphorylation inhibition. Our findings emphasize the high mobility of Arabidopsis SR splicing factors and provide insights into the dynamic relationships between the different nuclear compartments.