The RNA recognition motif (RRM) of the Arabidopsis RS2Z32 and RS2Z33 splicing factors coordinates protein–protein and protein–RNA interactions, and contributes to their nucleocytoplasmic dynamics

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. 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 to the establishment of protein-protein interactions and to 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. We demonstrate that their RNA recognition motif (RRM) domain specifically binds pyrimidine-rich RNA motifs via three residues (Y14, Y46, and 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 in the regulation of the splicing process.