Network of interactions of a novel plant-specific Arg/Ser-rich protein, atRSZ33, with atSC35-like splicing factors

Arg/Ser-rich (RS) proteins play a crucial role in splicing and are implicated in splice site selection in metazoa. In plants, intron recognition seems to differ from the one in animals due to specific factor requirements. Here we describe a new plant-specific RS-rich protein, atRSZ33, with a unique domain structure consisting of an RNA recognition motif (RRM), two zinc knuckles embedded in a basic RS region, and an acidic C-terminal domain. atRSZ33 was found to be a phosphoprotein that concentrates in nuclear speckles and is predominantly present in roots and flowers. In a yeast two-hybrid screen, atRSZ33 interacted with splicing factors atSRp34/SR1, an Arabidopsis ortholog of human SF2/ASF; atRSZp21 and atRSZp22, which are similar to the human 9G8; and three novel SC35-like splicing factors termed atSCL28, atSCL30, and atSCL33/SR33. Two further members of the SCL family, namely SCL30a and the ortholog of mammalian SC35, atSC35, were also found to interact with atRSZ33. These interactions were verified by in vitro binding assays; furthermore, the transcriptional activity of atRSZ33 was found to overlap with the ones of its interacting partners. These specific interactions coupled with the many similarities of atRSZ33 to SR proteins suggest that its main activity is in spliceosome assembly. Mapping of regions necessary for protein-protein interaction between atRSZ33 and atSCL33/SR33 revealed that both zinc knuckles together with a small part of the RS and the RRM domain are required for efficient binding. However, the interacting domain is relatively small, allowing binding of additional proteins, a feature that is consistent with the proposed role of atRSZ33 in spliceosome assembly.