An SC35-like protein and a novel serine/arginine-rich protein interact with Arabidopsis U1-70K protein

The U1 small nuclear ribonucleoprotein 70-kDa protein, a U1 small nuclear ribonucleoprotein-specific protein, has been shown to have multiple roles in nuclear precursor mRNA processing in animals. By using the C-terminal arginine-rich region of Arabidopsis U1-70K protein in the yeast two-hybrid system, we have identified an SC35-like (SR33) and a novel plant serine/arginine-rich (SR) protein (SR45) that interact with the plant U1-70K. The SR33 and SR45 proteins share several features with SR proteins including modular domains typical of splicing factors in the SR family of proteins. However, both plant SR proteins are rich in proline, and SR45, unlike most animal SR proteins, has two distinct arginine/serine-rich domains separated by an RNA recognition motif. By using coprecipitation assays we confirmed the interaction of plant U1-70K with SR33 and SR45 proteins. Furthermore, in vivo and in vitro protein-protein interaction experiments have shown that SR33 protein interacts with itself and with SR45 protein but not with two other members (SRZ21 and SRZ22) of the SR family that are known to interact with the Arabidopsis full-length U-70K only. A Clk/Sty protein kinase (AFC-2) from Arabidopsis phosphorylated four SR proteins (SR33, SR45, SRZ21, and SRZ22). Coprecipitation studies have confirmed the interaction of SR proteins with AFC2 kinase, and the interaction between AFC2 and SR33 is modulated by the phosphorylation status of these proteins. These and our previous results suggest that the plant U1-70K interacts with at least four distinct members of the SR family including SR45 with its two arginine/serine-rich domains, and the interaction between the SR proteins and AFC2 is modulated by phosphorylation. The interaction of plant U1-70K with a novel set of proteins suggests the early stages of spliceosome assembly, and intron recognition in plants is likely to be different from animals.     

Interaction of poly(A) polymerase with the 25-kDa subunit of cleavage factor I.

Mammalian poly(A) polymerase (PAP), a key enzyme in the pre-mRNA 3'-end processing reaction, carries the catalytic domain in the N-terminal region, an RNA binding domain, two nuclear localization signals, and a serine/threonine-rich regulatory domain in the C-terminal region. Using LexA-based yeast two-hybrid screening, we identified a cDNA encoding the 25-kDa subunit of cleavage factor I (CFI-25) as a protein that interacts with the C-terminal region of mouse PAP. The glutathione S-transferase pull-down assay and the immunoprecipitation experiment revealed that PAP directly interacts with CFI-25 and that the C-terminal 69 residues of PAP and the N-terminal 60 residues of CFI-25 are sufficient for the interaction between CFI-25 and PAP. Since CFI is known to function in the assembly of the pre-mRNA 3'-processing complex, this interaction may play an important role in the assembly of the processing complex and/or in the regulation of PAP activity within the complex.     

Arabidopsis thaliana PRP40s are RNA polymerase II C-terminal domain-associating proteins

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II functions as a scaffold for RNA processing machineries that recognize differentially phosphorylated conserved (YSPTSPS)_n repeats. Evidence indicates that proteins that regulate the phosphorylation status of the CTD are determinants of growth, development, and stress responses of plants; however, little is known about the mechanisms that translate the CTD phosphoarray into physiological outputs. We report the bioinformatic identification of a family of three phospho-CTD-associated proteins (PCAPs) in Arabidopsis and the characterization of the AtPRP40 (Arabidopsis thaliana PRE-mRNA-PROCESSING PROTEIN 40) family as PCAPs. AtPRP40s-CTD/CTD-PO₄ interactions were confirmed using the yeast two-hybrid assay and far-Western blotting. WW domains at the N-terminus of AtPRP40b mediate the AtPRP40b-CTD/CTD-PO₄ interaction. Although AtPRP40s interact with both phosphorylated and unphosphorylated CTD in vitro, there is a strong preference for the phosphorylated form in Arabidopsis cell extract. AtPRP40s are ubiquitously expressed and localize to the nucleus. These results establish that AtPRP40s are specific PCAPs, which is consistent with the predicted function of the AtPRP40 family in pre-mRNA splicing.     

The serine/arginine-rich protein family in rice plays important roles in constitutive and alternative splicing of pre-mRNA

Ser/Arg-rich (SR) proteins play important roles in the constitutive and alternative splicing of pre-mRNA. We isolated 20 rice (Oryza sativa) genes encoding SR proteins, of which six contain plant-specific characteristics. To determine whether SR proteins modulate splicing efficiency and alternative splicing of pre-mRNA in rice, we used transient assays in rice protoplasts by cotransformation of SR protein genes with the rice Waxy(b) (Wx(b))-beta-glucuronidase fusion gene. The results showed that plant-specific RSp29 and RSZp23, an SR protein homologous to human 9G8, enhanced splicing and altered the alternative 5' splice sites of Wx(b) intron 1. The resulting splicing pattern was unique to each SR protein; RSp29 stimulated splicing at the distal site, and RSZp23 enhanced splicing at the proximal site. Results of domain-swapping experiments between plant-specific RSp29 and SCL26, which is a homolog of human SC35, showed the importance of RNA recognition motif 1 and the Arg/Ser-rich (RS) domain for the enhancement of splicing efficiencies. Overexpression of plant-specific RSZ36 and SRp33b, a homolog of human ASF/SF2, in transgenic rice changed the alternative splicing patterns of their own pre-mRNAs and those of other SR proteins. These results show that SR proteins play important roles in constitutive and alternative splicing of rice pre-mRNA.     

The role of the coiled-coil motif in interactions mediated by TPD52

TPD52 (D52)-like proteins are small coiled-coil motif-bearing proteins first identified through their expression in human breast carcinoma that mutually interact in hetero- and homomeric fashions. However, it has been unclear whether the coiled-coil motif is sufficient, or even necessary, for these interactions to occur. We have therefore examined the binding activities of a panel of C-terminally deleted D52 proteins in both the yeast two-hybrid system and pull-down assays. In the yeast two-hybrid system, interactions were only detected when regions C-terminal to the coiled-coil motif were also present. However, using pull-down assays, interactions were detected for all deletion mutants which included the coiled-coil motif. This suggests that the coiled-coil motif is indeed necessary for interactions mediated by D52 proteins, but that C-terminal protein regions facilitate and/or stabilize these interactions.     

Ggamma13 interacts with PDZ domain-containing proteins

The G protein gamma13 subunit (Ggamma13) is expressed in taste and retinal and neuronal tissues and plays a key role in taste transduction. We identified PSD95, Veli-2, and other PDZ domain-containing proteins as binding partners for Ggamma13 by yeast two-hybrid and pull-down assays. In two-hybrid assays, Ggamma13 interacted specifically with the third PDZ domain of PSD95, the sole PDZ domain of Veli-2, and the third PDZ domain of SAP97, a PSD95-related protein. Ggamma13 did not interact with the other PDZ domains of PSD95. Coexpression of Ggamma13 with its Gbeta1 partner did not interfere with these two-hybrid interactions. The physical interaction of Ggamma13 with PSD95 in the cellular milieu was confirmed in pull-down assays following heterologous expression in HEK293 cells. The interaction of Ggamma13 with the PDZ domain of PSD95 was via the C-terminal CAAX tail of Ggamma13 (where AA indicates the aliphatic amino acid); alanine substitution of the CTAL sequence at the C terminus of Ggamma13 abolished its interactions with PSD95 in two-hybrid and pull-down assays. Veli-2 and SAP97 were identified in taste tissue and in Ggamma13-expressing taste cells. Coimmunoprecipitation of Ggamma13 and PSD95 from brain and of Ggamma13 and SAP97 from taste tissue indicates that Ggamma13 interacts with these proteins endogenously. This is the first demonstration that PDZ domain proteins interact with heterotrimeric G proteins via the CAAX tail of Ggamma subunits. The interaction of Ggamma13 with PDZ domain-containing proteins may provide a means to target particular Gbetagamma subunits to specific subcellular locations and/or macromolecular complexes involved in signaling pathways.