The Arabidopsis RS2Z32 and RS2Z33 proteins are dynamic splicing factors whose RNA recognition motif (RRM) domain contributes to protein-protein and protein-RNA interactions

Abstract

The Arabidopsis splicing factors arginine/serine-rich zinc knuckle-containing proteins 32 and 33 (RS2Z32 and RS2Z33) are plant-specific members of the SR family whose molecular functions received little attention. Here, we characterized both RS2Z32 and RS2Z33 by examining their expression profile at different stages of development and their spatial cellular distribution, as well as the contribution of their domains in the establishment of protein-protein interactions and RNA binding specificity. We report that the RS2Z32 and RS2Z33 promoters are ubiquitously active during vegetative and reproductive growth, and that both RS2Z splicing factors localize in the nucleus (except the nucleolus). We show that the C-terminal arginine/serine-rich (RS) domain, but not the serine/proline-rich (SP) extension, is a determinant of nuclear localization, which likely requires phosphoresidues putatively phosphorylated by kinases of the SRPK family. We demonstrate that their RNA recognition motif (RRM) domain specifically binds pyrimidine-rich RNA motifs via three residues (Y14, Y46, F48), and is also involved in protein-protein interactions with at least three SR proteins, namely SR45, SCL30, and SR34. Finally, we show that mutations in RNA-binding domains (i.e. RRM and zinc knuckles, ZnKs) affect the nucleocytoplasmic dynamics of both RS2Z proteins. Our findings provide molecular evidence for the involvement of plant-specific SR splicing factors into the regulation of the splicing process. HighlightSpecific domains of the Arabidopsis RS2Z splicing factors contribute to their nuclear localization, nucleocytoplasmic dynamics, and ability to contact protein partners and specific pyrimidine-rich RNA motifs.