ASAP, a novel protein complex involved in RNA processing and apoptosis

Abstract

Different isoforms of a protein complex termed the apoptosis- and splicing-associated protein (ASAP) were isolated from HeLa cell extract. ASAP complexes are composed of the polypeptides SAP18 and RNPS1 and different isoforms of the Acinus protein. While Acinus had previously been implicated in apoptosis and was recently identified as a component of the spliceosome, RNPS1 has been described as a general activator of RNA processing. Addition of ASAP isoforms to in vitro splicing reactions inhibits RNA processing mediated by ASF/SF2, by SC35, or by RNPS1. Additionally, microinjection of ASAP complexes into mammalian cells resulted in acceleration of cell death. Importantly, after induction of apoptosis the ASAP complex disassembles. Taken together, our results suggest an important role for the ASAP complexes in linking RNA processing and apoptosis.

FIG. 1.

Purification of a complex consisting of SAP18, RNPS1, and Acinus-L. (a) Schematic representation of Acinus isoforms. Described protein domains as well as protease cleavage sites and their corresponding amino acids (1, 30) are indicated as follows: red box, P-loop for nucleotide binding; green box, SAP domain; dark blue box, region similar to RNA recognition motif of Drosophila Sxl; black box, region specific for Acinus-S; black arrowhead, cleavage site for unknown protease; red arrowhead, cleavage site for caspase 3. Regions recognized by polyclonal antibodies against Acinus ([N], [S(N10)], [A19], [M], and [C]) (see also Materials and Methods) are shown above the representation of Acinus-L. (b) Purification scheme used to isolate the ASAP complexes from HeLa cell nuclear pellets. (c) Silver stain of an SDS-4 to 15% (wt/vol) polyacrylamide gradient gel containing aliquots of fractions (input [I] and the indicated column fractions) derived from the MonoP column. High-molecular-mass standards [M] are indicated at the left, and components of the ASAP complex are indicated at the right side of the panel. A putative alternatively phosphorylated or partially degraded form of RNPS1 is marked with an asterisk. (d) Western blot analysis of the samples used in panel c. The antibodies used in the Western blot are indicated at the right, and high-molecular-mass standards [M] are indicated at the left side of the panel. (e) Verification of the complex composition by coimmunoprecipitation. Antibodies against SAP18 (lane 5) but not MTA-2 (lane 4) coimmunoprecipitate RNPS1 and Acinus-L from a protein fraction derived from nuclear pellet [In]. Samples in lanes 2 to 5 were treated with alkaline phosphatase [AP] after immunoprecipitation in an attempt to increase the signal for Acinus-L by focusing putative differently phosphorylated species. Ten percent of the input is loaded in lanes 1 and 2. Immunoprecipitated proteins were separated on an SDS-4 to 15% (wt/vol) polyacrylamide gradient gel and analyzed by Western blotting with the antibodies indicated at the right. Lanes labeled [M] contain high-molecular-mass protein standards.

FIG. 2.

A second ASAP complex containing Acinus-S. (a) Silver stain of an SDS-4 to 15% (wt/vol) polyacrylamide gradient gel containing aliquots of the indicated fractions derived from the final MonoP column. Components of the ASAP-S complex are indicated at the right side of the panel. A putative alternatively phosphorylated or partially degraded form of RNPS1 is marked with an asterisk. HMW, higher-molecular-weight bands. (b) Western blot analysis of fraction 33 of the MonoP column (lanes 2, 4, 6, 8, and 10). The Acinus antibodies used in the Western blot are indicated at the top (compare with Fig. 1a). High-molecular-mass protein standards [M] (lanes 1, 3, 5, 7, and 9) are indicated at the left.

FIG. 3.

Localization of ASAP complex components in HeLa cells. (a) HeLa cells were transiently transfected with a Flag-RNPS1 expression construct. Overexpression of Flag-RNPS1 leads to formation of a few large mega-speckles (left panel, arrowheads) or more abundant and smaller speckles (right panel). Localization of Flag-RNPS1 and SAP18 was analyzed by indirect immunofluorescence with antibodies against the Flag tag and SAP18. SAP18 is present in multiple nuclear sites in untransfected cells (left panel, arrows) but colocalizes with Flag-RNPS1 in transfected cells. (b) HeLa cells were transfected and analyzed with antibodies against the Flag tag and Acinus as in panel a. Mega-speckles caused by overexpression of Flag-RNPS1 are indicated by arrowheads, and localization of Acinus in nuclear speckles in untransfected cells is indicated by arrows. Acinus colocalizes with Flag-RNPS1 in transfected cells. (c) HeLa cells transiently transfected with Flag-RNPS1 were analyzed with a fluorescein isothiocyanate-coupled monoclonal antibody against the Flag tag and by indirect immunofluorescence with an antibody against Sin3. Sin3 shows nuclear staining and does not colocalize with Flag-RNPS1 in the mega-speckles. (d) Untransfected HeLa cells were analyzed for localization of SAP18 and Acinus by indirect immunofluorescence with antibodies against SAP18 and the carboxyl terminus of Acinus, respectively. SAP18 and Acinus colocalize in nuclear speckles. (e) Untransfected HeLa cells were analyzed as in panel d with antibodies against SAP18 and SC35, respectively. SAP18 localizes to nuclear speckles containing SC35.

FIG. 4.

ASAP-L complex inhibits RNPS1-activated or ASF/SF2-mediated splicing in S100 extracts. Increasing amounts of purified ASAP-L complex (80 and 320 ng, 1.25 and 5 μl; lanes 3, 4, 10, and 11) or recombinant SAP18 (3, 9, and 27 ng; lanes 5 to 7 and 12 to 14) were added to S100 extracts complemented with limiting (3 ng; lanes 1 to 7) or optimal (40 ng; lanes 8 to 14) amounts of ASF/SF2 in the presence of RNPS1 (360 ng; lanes 2 to 7). Lane 1, S100 plus 3 ng of ASF/SF2 only; lane 2, S100 plus 3 ng of ASF/SF2 and 360 ng of RNPS1; lane 8, S100 plus 40 ng of ASF/SF2 only; lane 9, S100 plus 40 ng of ASF/SF2 and 360 ng of RNPS1. As determined by Western blot analyses (data not shown), 1.5 μl of purified ASAP-L contained amounts of recombinant RNPS1 and SAP18, which were stoichiometric to 14.4 ng of RNPS1 and 6.5 ng of SAP18, respectively. RNA was fractionated on a denaturing polyacrylamide gel, and splicing products, indicated schematically on the right, were visualized by autoradiography.

FIG. 5.

Analysis of ASAP-S, Acinus-L, and SAP18 in the in vitro splicing assay. Increasing amounts of purified ASAP-L (1.25 and 5 μl; lanes 5, 6, 15, and 16), ASAP-S (0.875 μl and 3.5 μl; lanes 7, 8, 17, and 18), and baculovirus-expressed recombinant Acinus-L (30, 90, and 260 ng; lanes 2, 3, 4, 12, 13, and 14) or SAP18 (4 and 12 ng; lanes 9, 10, 19, and 20) were added to S100 extracts complemented with limiting (5 ng; lanes 1 to 10) or optimal (50 ng; lanes 11 to 20) amounts of ASF/SF2. Lane 1, S100 plus 5 ng of ASF/SF2 only; lane 11, S100 plus 50 ng of ASF/SF2 only. As determined by Western blot analyses (data not shown), 5 μl of purified ASAP-L contained amounts of Acinus-L and SAP18, which were stoichiometric to 260 ng of recombinant Acinus-L and 12 ng of recombinant SAP18, respectively. Purified ASAP-S (3.5 μl) contained stoichiometric amounts of ASAP to 5 μl of ASAP-L. RNA was fractionated on a denaturing polyacrylamide gel, and splicing products, indicated schematically on the right, were visualized by autoradiography.

FIG. 6.

Function of ASAP during apoptosis. (a) Microinjection of ASAP complexes into HeLa cells. HeLa cells were microinjected with a caspase 8 expression construct (purple line), highly purified ASAP complexes (ASAP-L, blue line; ASAP-S, red line), or with buffer only (control, orange line). After injection the cells were allowed to recover for >2 h at 37°C in 7% CO₂. Cell death was subsequently induced with 1 μM staurosporine. Apoptosis was determined on the basis of morphological criteria at the indicated time points. Equimolar amounts (determined by Western blot analyses; data not shown) of ASAP-L and ASAP-S were injected. Experiments were performed in triplicate, and standard deviations are shown. (b) Western blot analysis of the immunoprecipitates obtained with antibodies against SAP18. Proteins were immunoprecipitated from extracts prepared from HeLa cells, which were either untreated (−; lanes 2 and 5) or treated with 5 μM staurosporine for 8 h (+; lanes 3 and 6). Precipitated proteins are loaded in lanes 5 and 6, and 10% of the input is loaded in lanes 2 and 3. Immunoprecipitates were analyzed as in Fig. 1e, with the antibodies indicated at the right. Lanes labeled M contain high-molecular-mass protein standards (lanes 1 and 4).