Tra2-beta1 binds to DARPP-32 in vivo
We previously showed that tra2-beta1 binds protein phosphatase 1 (PP1) via an evolutionary conserved PVXF motif in the RNA recognition motif of tra2-beta1 [8
]. These findings suggested that PP1 is present in a complex with splicing factors. We next asked whether other PP1 interacting proteins could also interfere with splicing regulatory proteins. Unexpectedly, during these experiments we found that purified DARPP-32, a known PP1 interactor, binds to tra2-beta1. This was surprising, as a DARPP32:tra2-beta1 interaction has not been detected previously in yeast-two hybrid assays [6
]. We next confirmed these results by co-immunoprecipitation experiments and determined which protein domains are necessary for the interaction. Tra2-beta1 is composed of a central RNA binding motif flanked by two RS domains (). The RS domains are characterized by clusters of arginine-serine repeats and allow interaction between SR proteins. The RRM of tra2-beta1 binds to RNA and PP1. To investigate which tra2-beta1 protein parts are responsible for binding to DARPP-32 we performed immunoprecipitation experiments using tra2-beta1 variants lacking the first, second or both RS domains. As shown in , deletion of the first RS domain has no effect on DARPP-32 binding, whereas deletion of the second RS domain prevents binding.
Tra2-beta1 binds to DARPP-32 in vivo
To determine whether DARPP-32 can interact with endogenous tra2-beta1, we analyzed immunoprecipitates made from HEK239 cells that transiently express EGFP-DARPP-32. As shown in , we could detect endogenous tra2-beta1 in these immunoprecipitates, suggesting that the interaction can take place under physiological tra2-beta1 concentrations.
We next ask whether other SR-proteins could interact with DARPP-32 and coexpressed EGFP-tagged DARPP-32 with Flag-tagged SRp30c, SF2/ASF and SC35. As shown in , we could not detect these SR-proteins in the DARPP-32 immunoprecipitates.
Together, this data indicate that DARPP-32 binds to tra2-beta1 and that the second RS domain of tra2-beta1 is necessary for DARPP-32 binding. In contrast to tra2-beta1, several other SR-proteins do not interact with DARPP-32.
DARPP-32 colocalizes with tra2-beta1 in the nucleus of primary neurons
To investigate whether the interaction between DARPP-32 and tra2-beta1 could occur in cells, we performed double immunohistochemistry staining experiments using primary neuronal cultures. We used hippocampal neurons, which express low levels of DARPP-32 in vivo, but in which we found a significant labeling in culture. Neurons derived from E18 embryos were stained for endogenous DARPP-32 and tra2-beta1 after 14 days of culture. As shown in both proteins show an overlapping staining in the nucleus. To determine whether there is a colocalisation in another cell type, we tested the localization of overexpressed DARPP-32 and endogenous tra2-beta1 in HEK293 cells. As shown in , in these cells DARPP-32 is primarily cytosolic, but can also be found in the nucleus, where DARPP-32 colocalizes with tra2-beta1. No staining was observed when the primary antisera were omitted (). Together, these results argue that both proteins colocalize in vivo and could indeed interact with each other.
tra2-beta1 colocalizes with DARPP-32
PP1 antagonizes the binding of DARPP-32 to tra2-beta1
We next tested whether the interaction between DARPP-32 and tra2-beta1 is direct and performed pull-down experiments using purified proteins. His-tra2-beta1 generated in insect cells [8
] was coupled to Ni-agarose. This affinity matrix was incubated with purified DARPP-32. To rule out nucleic acid mediated interaction, benzonase was present in the binding reaction. The protein mixture was washed three times in Native Wash Buffer. After washing, protein was removed by boiling in 1% SDS and analyzed by PAGE followed by Western blot. As shown in , lane 4, this affinity resin bound DARPP-32. In contrast, no DARPP-32 was retained when no tra2-beta1 was prior bound to the resin (, lane 6). This experiment supports the interaction indicated by immunoprecipitation and shows that the binding between DARPP-32 and tra2-beta1 is based on direct protein:protein interaction.
DARPP-32 binds directly to tra2-beta1 and antagonizes PP1 binding to tra2-beta1
PP1 binds to both tra2-beta1 and DARPP-32 ([8
], , ). To test whether both proteins compete for PP1 binding, we incubated both PP1 and DARPP-32 with the tra2-beta1-affinity column. We found that the presence of PP1 reduced the amount of DARPP-32 bound to the column, (, lane 5). To further test whether DARPP-32 and PP1 compete for tra2-beta1 binding, we used different PP1 concentrations. The tra2-beta1 affinity matrix was loaded with purified DARPP-32 (50 μM), and subsequently half and equal molar amounts of PP1 were added. As shown in , lane 2 and 3, the added PP1 replaced DARPP-32 bound to tra2-beta1. Finally, we tested whether the PP1 binding site in the RRM of tra2-beta1 is involved in DARPP-32 binding and used tra2-beta1-RATA protein as an interacting partner. In this protein, the PP1 binding site in the beta4 strand of the RRM is changed from RVDF to RATA, which completely abolished PP1 binding [8
]. As shown in , lane 4, DARPP-32 binds to tra2-beta1-RATA, indicating the PP1 binding site is not involved in DARPP-32 binding. Together, these data indicate that PP1 competes with DARPP-32 to bind to tra2-beta1.
DARPP-32 overexpression changes alternative splicing of reporter genes
DARPP-32 changes alternative splicing of tra2-beta1 dependent exons
Tra2-beta1 binds to GAA-rich sequences and generally promotes inclusion of alternative exons that contain such motifs [7
]. We therefore determined whether DARPP-32 influences alternative splice site selection of two of such exons, the alternatively spliced exon 2 of the tra2-beta1 pre-mRNA and the tau exon 10 [7
]. First, we transfected an increasing amount of DARPP-32 expression construct together with splicing reporter genes into HEK293 cells. As shown in , an increased amount of DARPP-32 caused skipping of the alternative exon in both the tau and tra2-beta1 reporter constructs, that paralleled the increase of DARPP-32 protein caused by the overexpression ().
We next performed the reverse experiment and removed DARPP-32 by siRNA treatment. As shown in , siRNA treatment promoted inclusion of both alternative exons, which again paralleled the decrease of DARPP-32 caused by siRNA treatment.
siRNA mediated DARPP-32 knock down changes alternative splicing of reporter genes
These experiment indicates that the relative concentration of DARPP-32 influences alternative splice site selection.