Here we report the purification of a novel protein complex composed of the polypeptides SAP18, RNPS1, and Acinus. We chose the name ASAP complex because the subunit composition and our functional analyses suggest that the complex participates in both apoptosis and RNA splicing. The ASAP complexes exists in at least two isoforms, termed ASAP-L and ASAP-S, which are characterized by the presence of different isoforms of Acinus, and members of the ASAP complex have been identified in nuclear compartments thought to be involved in RNA processing (N. Saitoh and D. L. Spector, personal communication).
While SAP18 has not previously been implicated in pre-mRNA splicing, an involvement in RNA processing was suggested for both Acinus and RNPS1, which were purified as components of functional spliceosomes (29
). Furthermore, RNPS1 has been shown to localize to nuclear splicing factor compartments (19
) and was purified as a single-subunit protein that mediates the general activation of splicing that occurs in the presence of limited amounts of SR proteins in an in vitro splicing assay (25
). In the context of the ASAP complex, this positive effect on RNA processing in vitro is suppressed (Fig. and ). Repression of RNA processing most likely occurs at an early stage of the splicing reaction. Therefore, ASAP inhibits splicing under conditions where RNPS1 alone can function with SR proteins to activate splicing, indicating that incorporation of RNPS1 into the ASAP complexes can serve to regulate the splicing activity of RNPS1. Modification of ASAP complex components might also modulate the activity of ASAP during RNA processing.
Several recent reports have presented evidence of a role for RNPS1 in downstream events of mRNA metabolism (16
). In the current model, RNPS1 binds to RNA in the nucleus as part of a postsplicing complex deposited upstream of exon-exon junctions, which is thought to provide a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay. RNPS1 in this exon junction complex travels with the RNA into the cytoplasm and communicates the position of exon-exon junctions for the mRNA surveillance machinery (20
). It is unclear whether the fraction of RNPS1 incorporated in the ASAP complexes might play a similar role or if formation of the ASAP complex functions to sequester RNPS1, preventing it from functioning in this capacity.
Acinus had previously been implicated to function during apoptotic chromatin condensation (30
). Microinjection analyses of the ASAP complexes clearly demonstrate that ASAP influences the progression of apoptotic cell death (Fig. ). Modulation of cell death by ASAP-L also occurred when apoptosis was induced by a combination of tumor necrosis factor α and cycloheximide (data not shown), suggesting that the effects caused by ASAP are at least partly direct. Various studies have provided evidence that SAP18 can associate with the Sin3-HDAC complex (4
). Considering the function of ASAP in execution of apoptosis and the potential role of Acinus in apoptotic chromatin condensation, these observations raise the exciting possibility that deacetylation of histones via recruitment of the Sin3-HDAC complex is involved in the process of chromatin condensation during apoptosis. However, we have not yet been able to demonstrate association of ASAP and Sin3-HDAC complexes. It is noteworthy that in the above-mentioned studies (4
), demonstration of interaction of SAP18 with the Sin3-HDAC complex required overexpression of SAP18, suggesting that just a small fraction of cellular SAP18 associates with the Sin3-HDAC complex. This is in agreement with our observations that although SAP18 is present in several multiprotein complexes of different sizes, the majority of SAP18 copurifies with RNPS1 and different Acinus isoforms (data not shown). To clarify a putative role of the Sin3-HDAC complex during apoptotic chromatin condensation, further experimentation is required.
Since the Acinus subunit of the ASAP complex is a target of proteolytic cleavage during apoptosis (30
), it is possible that apoptotic stimuli lead to a regulation of the function of the complex in splicing through reorganization of the complex composition. Regulation of splicing has been shown to play an important role during apoptosis (3
). IP analysis of extracts derived from apoptotic cells strongly indicates that the complex disassembles during cell death (Fig. ). If activation of splicing by RNPS1 is repressed by incorporation into ASAP, this event would trigger release of the activation function of that fraction of RNPS1 in splicing, thereby modulating RNA processing of possibly selected target transcripts. Interestingly, a link between splicing and apoptosis is established not only by the Acinus protein but also by RNPS1. RNPS1 has been shown to interact with the 110-kDa isoform of the p34cdc2
-related protein kinase PITSLRE (p110). p110 is processed during apoptosis into a smaller isoform(s) that does not interact with RNPS1 (19
), and a recently identified cyclin, cyclin L, interacts with p110 (2
). The carboxyl terminus of this particular cyclin contains numerous arginine-serine-rich dipeptide repeats, characteristic of many splicing factors, and may target the p110/cyclin L complex to nuclear speckles. Antibodies against cyclin L inhibit RNA processing in an in vitro assay, and recombinant cyclin L stimulates splicing under suboptimal conditions (5
). A smaller cyclin L protein is also made by alternative splicing, which retains the cyclin box region but no longer contains the RS domain. However, the caspase-processed p46 PITSLRE isoform interacts well in vivo only with the smaller form of cyclin L (J. H. Trembley, D. Hu, and V. J. Kidd, unpublished data). Further experimentation is required to decipher whether there is a functional interplay between the ASAP complex, PITSLRE isoforms, and cyclin L or L short forms during apoptosis.
In summary, we have identified a novel protein complex (ASAP complex) consisting of SAP18, RNPS1, and Acinus. Functional analyses suggest that ASAP is involved in both RNA processing and apoptosis, pointing to ASAP as a reasonable candidate for participation in the regulation of splicing during the execution of programmed cell death.