Chemokine (C-C Motif) Ligand 2 Engages CCR2+ Stromal Cells of Monocytic Origin to Promote Breast Cancer Metastasis to Lung and Bone

Metastatic spread of cancer to distant vital organs, including lung and bone, is the overwhelming cause of breast cancer mortality and morbidity. Effective treatment of systemic metastasis relies on the identification and functional characterization of metastasis mediators to multiple organs. Overexpression of the chemokine (C-C motif) ligand 2 (CCL2) is frequently associated with advanced tumor stage and metastatic relapse in breast cancer. However, the functional mechanism of CCL2 in promoting organ-specific metastasis of breast cancer has not been rigorously investigated. Here, we used organ-specific metastatic sublines of the MDA-MB-231 human breast cancer cell line to demonstrate that overexpression of CCL2 promotes breast cancer metastasis to both lung and bone. Conversely, blocking CCL2 function with a neutralizing antibody reduced lung and bone metastases. The enhancement of lung and bone metastases by CCL2 was associated with increased macrophage infiltration and osteoclast differentiation, respectively. By performing functional assays with primary cells isolated from the wild type, CCL2 and CCR2 knock-out mice, we showed that tumor cell-derived CCL2 depends on its receptor CCR2 (chemokine, CC motif, receptor 2) expressed on stromal cells to exert its function in promoting macrophage recruitment and osteoclast differentiation. Overall, these data demonstrated that CCL2-expressing breast tumor cells engage CCR2⁺ stromal cells of monocytic origin, including macrophages and preosteoclasts, to facilitate colonization in lung and bone. Therefore, CCL2 and CCR2 are promising therapeutic targets for simultaneously inhibiting lung and bone metastasis of breast cancer.Breast cancer is the most common malignancy in women in the United States, with an estimated 182,000 new cases and 40,000 deaths in 2008 (1). Late stage breast cancer patients develop metastases in bone, lung, liver, brain, and other organs, which are responsible for most breast cancer-related mortality and morbidity (2). Severe complications from bone metastasis include debilitating bone fractures, nerve compression and bone pain, and hypercalcemia (3–5), whereas lung metastasis is accompanied by cough, bloody sputum, rib cage pain, and, eventually, failure of the respiratory functions (6). Colonization of different secondary organs by breast cancer is believed to be a complex, multigenic process that depends on productive interactions between tumor cells and stromal microenvironments through concerted actions of organ-specific metastasis genes (7, 8). Functional genomic analysis of preclinical models of breast cancer to bone, lung, and brain have identified distinct sets of organ-specific metastasis genes (9–11), providing novel mechanistic insights into key rate-limiting steps of metastasis to different organs. However, as advanced breast cancer patients often suffer from metastases at several secondary organs, identifying genes that are capable of instigating metastasis to multiple sites may provide the ideal targets for therapeutic intervention of systemic metastasis.Chemokines are small (8–14 kDa) proteins classified into four conserved groups (CXC, CC, C, and CX3C) based on the position of the first two cysteines that are adjacent to the amino terminus (12). They are chemotactic cytokines that stimulate directed migration of leukocytes in response to inflammatory signals. Chemokines are also involved in the maintenance of hematopoietic homeostasis, regulation of cell proliferation, tissue morphogenesis, and angiogenesis (13). Chemokines bind to the seven-transmembrane domain receptors to elicit downstream molecular events that coordinate cell movement. Even though chemokines are unlikely to be a contributing factor for tumor initiation, they can have pleiotropic effects on tumor progression (13, 14). Among more than 50 human chemokines, CCL2 is of particular importance. CCL2, also called monocyte chemoattractant protein-1 (MCP-1), is a potent chemoattractant for monocytes, memory T lymphocytes, and natural killer cells (15). It is involved in a number of inflammatory conditions associated with monocyte recruitment, including delayed hypersensitivity reactions, bacterial infection, arthritis, and renal disease (15). The importance of CCL2 in cancer was manifested by its overexpression in a variety of tumor types, including glioma, ovarian, esophagus, lung, breast, and prostate cancers (15–17). In prostate cancer, CCL2 expression levels was associated with advanced pathological stage (16). Importantly, CCL2-neutralizing antibodies inhibit bone resorption in vitro and bone metastasis in vivo (18–20). In lung cancer, serum CCL2 levels were elevated in lung cancer patients with bone metastasis compared with localized diseases. Neutralizing antibodies against CCL2 also inhibited the tumor conditioned media-induced osteoclast formation in vitro and bone metastasis in vivo (17). Taken together, these findings suggested a role of CCL2 in bone metastasis.A potential role of CCL2 in breast cancer progression and metastasis has been indicated by the analysis of CCL2 expression of tumor and serum samples from breast cancer patients. Serum CCL2 levels were significantly higher in postmenopausal breast cancer patients than in age-matched controls (21). Over 50% of breast cancer tumor samples had intense staining of CCL2 in tumor cells (22). Prognostic analysis further revealed that high expression of CCL2 was correlated with advanced tumor stage, lymph node metastasis (23), and early relapse (24). CCL2 up-regulation in breast tumors was also associated with the infiltration of tissue-associated macrophages (TAMs)³ and with increased microvessel density (22, 24). TAMs have been known to contribute to primary tumor progression and metastasis of breast cancer (25), which is supported by epidemiological evidence showing that TAM infiltration portended a poor clinical outcome (26, 27). However, whether the function of CCL2 in modulating activity of macrophages and possibly other cell types is important for breast tumor organotropic metastasis has not been rigorously investigated. CCL2 may engage organ-specific cell types derived from the same bone marrow myelomonocytic progenitors. These progenitors differentiate into osteoclast precursors in bone or into blood monocytes that eventually become mature macrophages in different tissues, like alveolar macrophages in lung (28). These stromal cell types of myelomonocytic origin may contribute to different functions in different organ-specific metastases. Another unresolved question regarding the function of CCL2 in tumor-stroma interaction is the functional involvement of CCL2 receptors. CCL2 can bind to both CCR2 and CCR4 (29, 30). Loss of function studies in mice showed CCL2 and CCR2 knock-out mice displayed similar impairments in monocyte migration (31, 32), suggesting that CCR2 is the major functional receptor for CCL2. Understanding whether CCR2 deficiency in stromal cells leads to compromised monocyte engagement by CCL2-expressing tumor cells may have important implications in designing targeting therapeutics against the CCL2/CCR2 axis.In this study, we used the recently developed organ-specific metastatic sublines of the human breast cancer cell MDA-MB-231 (9, 10, 33) and showed that overexpression of CCL2 promotes both lung and bone metastases. This function was associated with increased TAM infiltration in lung metastasis and increased osteoclast differentiation in bone metastasis, respectively. Furthermore, by using macrophages and bone marrow cells isolated from wild type, CCL2-deficient, and CCR2-deficient mice, we showed that CCR2 expression in stromal cells is essential for tumor-derived CCL2 to recruit macrophages and promote osteoclastogenesis. Targeting tumor-derived CCL2 by a neutralizing antibody significantly reduced metastasis formation in both bone and lung.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Activin A Suppresses Osteoblast Mineralization Capacity by Altering Extracellular Matrix (ECM) Composition and Impairing Matrix Vesicle (MV) Production

During bone formation, osteoblasts deposit an extracellular matrix (ECM) that is mineralized via a process involving production and secretion of highly specialized matrix vesicles (MVs). Activin A, a transforming growth factor-β (TGF-β) superfamily member, was previously shown to have inhibitory effects in human bone formation models through unclear mechanisms. We investigated these mechanisms elicited by activin A during in vitro osteogenic differentiation of human mesenchymal stem cells (hMSC). Activin A inhibition of ECM mineralization coincided with a strong decline in alkaline phosphatase (ALP¹) activity in extracellular compartments, ECM and matrix vesicles. SILAC-based quantitative proteomics disclosed intricate protein composition alterations in the activin A ECM, including changed expression of collagen XII, osteonectin and several cytoskeleton-binding proteins. Moreover, in activin A osteoblasts matrix vesicle production was deficient containing very low expression of annexin proteins. ECM enhanced human mesenchymal stem cell osteogenic development and mineralization. This osteogenic enhancement was significantly decreased when human mesenchymal stem cells were cultured on ECM produced under activin A treatment. These findings demonstrate that activin A targets the ECM maturation phase of osteoblast differentiation resulting ultimately in the inhibition of mineralization. ECM proteins modulated by activin A are not only determinant for bone mineralization but also possess osteoinductive properties that are relevant for bone tissue regeneration.The quality of bone tissue is determined by the balanced action of the anabolic bone cells, the osteoblasts, and their catabolic counterparts, the osteoclasts. This process of bone remodeling occurs throughout life and can be influenced by a wide variety of molecules, having ultimately an impact on the quality of bone (1, 2). Activins and inhibins are members of the TGF-β superfamily with predominant antagonistic effects in their classically known target tissues, such as in gonadotropin producing cells in the pituitary and their role in reproduction (3, 4). Like other TGF-β member, activins elicit biological responses by binding to type I and II serine/threonine kinase receptors at the cell surface. Upon ligand binding, signaling is further transduced in the cytoplasm by phosphorylated Smad protein complexes that once in the nucleus regulate gene expression. This signaling pathway is highly complex because of crosstalk between different ligands (Activins, BMPs, TGF-β) binding to multiple serine/threonine kinase receptors that activate different Smad proteins signaling to the nucleus. Activin is known to signal using type II receptors ACVR2A or ACVR2B and the type I receptor ACVRIB (shared with BMPs) activating Smad2 and 3 proteins (shared with TGF-β). Inhibins exert their inhibitory effects on activin by competitive binding to the activin receptors in the presence of betaglycan. This signaling regulates a wide array of biological activities from cell proliferation, differentiation to tumor development and endocrine signaling (5, 6) in many cell lineages like hematopoietic (7, 8) and monocyte/macrophage (9, 10). Several consequences of these reproductive hormones, especially those of activin A, are also described in relation to bone metabolism. Activin A is present in bone tissue (11, 12) affecting both osteoclasts and osteoblasts. While having a consistent pro-osteoclastogenic effect (9, 13), the activin A impact on osteoblast differentiation is more controversial (see (14) for review) Several reports support a stimulatory effect of activin A on osteoblast differentiation and mineralization in vitro and in vivo (9, 15, 16). On the other hand, two different studies, using rat and human bone formation models, have demonstrated that activin A treatment has a coherent inhibitory influence on osteogenesis leading to significant reduction of the mineralization capacity (11, 17). These opposing effects of activin A on osteoblastogenesis may simply reflect species differences, however, it may be also driven by heterogeneity of the used cell model or the stage of osteoblast differentiation (14). Nevertheless, a negative role of activin A in bone formation is also supported by other in vivo studies in mice and primates in which blockage of activin signaling resulted in increased bone mass (18, 19). Moreover, transgenic mice overexpressing human inhibin A showed increased bone formation (20).The extracellular compartment is crucial for bone because it determines most of the bone quality properties (21, 22), including its strength, stability, and integrity. Interestingly, a mature extracellular matrix (ECM) is characterized by the capacity to mineralize even in the absence of further osteoblast activity (11, 23). This biomineralization process is complex and not fully elucidated but it is thought to be started within MVs (24). Osteoblasts in bone and other cells in mineralization competent tissues, such as cartilage (25), tendon (26), teeth (27), and calcifying vasculature (28) produce and release from their plasma membrane these vesicles with diameters ranging between 50 and 200 nm. It is inside these membrane-enclosed particles that first crystals of mineral are formed and grow, before the vesicle membrane is permeated and the mineral crystallization advances into the ECM (29, 30). In this context, proteins that can mobilize calcium and inorganic phosphate (Pi), the backbone of the hydroxyapatite crystals present in bone, are of utmost importance. P_i donor proteins found in MVs include alkaline phosphatase (ALP) and inorganic pyrophosphatases (31) whereas the annexin family of proteins is postulated to be crucial for calcium influx into the vesicles (32–34).In this study we investigated the inhibitory effect of activin A on human mesenchymal stem cells (hMSC) derived osteoblast differentiation and mineralization. We have previously shown that in human osteoblast cultures activin A influences the expression of many ECM genes altering ECM maturity (11). Thus, we focused our analysis on extracellular environment changes, namely the ECM and matrix vesicles (MVs). The characterization of these compartments was done using the state-of-the-art quantitative proteomics tools including SILAC metabolic labeling and mass spectrometry. Furthermore, the importance of ECM composition for osteoblast differentiation was also determined.     

Targeting Synaptic Pathology with a Novel Affinity Mass Spectrometry Approach

We report a novel strategy for studying synaptic pathology by concurrently measuring levels of four SNARE complex proteins from individual brain tissue samples. This method combines affinity purification and mass spectrometry and can be applied directly for studies of SNARE complex proteins in multiple species or modified to target other key elements in neuronal function. We use the technique to demonstrate altered levels of presynaptic proteins in Alzheimer disease patients and prion-infected mice.One prominent pathological feature of neuropsychiatric disorders such as Alzheimer disease (AD)¹ is severe synaptic loss (1–3). Previous reports of AD patients have shown that presynaptic dysfunction might occur early in the disease process (1, 4). Cortical synapse pathology has also been shown to correlate to the severity of dementia more closely than other pathological hallmarks of AD such as plaques and neurofibrillary tangles (5, 6). The SNARE proteins are essential components for the regulation of neurotransmitter exocytosis at the presynaptic site (7). Animal models suggest that changed expression or modification of SNARE complex proteins (synaptosomal-associated protein 25 (SNAP-25), syntaxin-1, and vesicle-associated membrane protein (VAMP)) alters synaptic function and is an interesting target for the development of therapeutics for neuropsychiatric illness (8, 9). The constituents of the SNARE complex are either localized in synaptic vesicles (VAMPs) or anchored at the presynaptic plasma membrane (SNAP-25 and syntaxin). The SNARE proteins are tightly assembled, and subsequent neurotransmitter release of the complex is quickly dissociated by N-ethylmaleimide-sensitive factor (7, 10–12). Because they are both strongly associated into complexes and membrane associated, the SNARE proteins are difficult to analyze via mass spectrometry, which is incompatible with most detergents necessary for the solubilization of proteins. Each SNARE complex protein exists in several isoforms that are differently distributed within the central nervous system (13–18). Post-translational modifications and truncated variants of the SNARE proteins make investigation of the protein expression even more complicated.In this study we developed an approach for the characterization and concurrent quantification of SNARE complex proteins that combines affinity purification by immunoprecipitation and mass spectrometry (IP-MS). We used precipitation with monoclonal antibodies against SNAP-25 to target the SNARE complex proteins and nanoflow LC–tandem mass spectrometry (LC-MS/MS) to characterize the co-immunoprecipitated interaction partners. Selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer coupled to a microflow LC system was used for quantification of the SNARE proteins. To demonstrate the usability of the IP-MS method, we performed a comparison of SNARE complex protein levels in brain tissue from AD patients and age-matched controls, as well as a study of SNARE complex protein levels in brain tissue from prion-infected mice.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Identification of Tumor-associated Autoantigens for the Diagnosis of Colorectal Cancer in Serum Using High Density Protein Microarrays

There is a mounting evidence of the existence of autoantibodies associated to cancer progression. Antibodies are the target of choice for serum screening because of their stability and suitability for sensitive immunoassays. By using commercial protein microarrays containing 8000 human proteins, we examined 20 sera from colorectal cancer (CRC) patients and healthy subjects to identify autoantibody patterns and associated antigens. Forty-three proteins were differentially recognized by tumoral and reference sera (p value <0.04) in the protein microarrays. Five immunoreactive antigens, PIM1, MAPKAPK3, STK4, SRC, and FGFR4, showed the highest prevalence in cancer samples, whereas ACVR2B was more abundant in normal sera. Three of them, PIM1, MAPKAPK3, and ACVR2B, were used for further validation. A significant increase in the expression level of these antigens on CRC cell lines and colonic mucosa was confirmed by immunoblotting and immunohistochemistry on tissue microarrays. A diagnostic ELISA based on the combination of MAPKAPK3 and ACVR2B proteins yielded specificity and sensitivity values of 73.9 and 83.3% (area under the curve, 0.85), respectively, for CRC discrimination after using an independent sample set containing 94 sera representative of different stages of progression and control subjects. In summary, these studies confirmed the presence of specific autoantibodies for CRC and revealed new individual markers of disease (PIM1, MAPKAPK3, and ACVR2B) with the potential to diagnose CRC with higher specificity and sensitivity than previously reported serum biomarkers.Colorectal cancer (CRC)¹ is the second most prevalent cancer in the western world. The development of this disease takes decades and involves multiple genetic events. CRC remains a major cause of mortality in developed countries because most of the patients are diagnosed at advanced stages because of the reluctance to use highly invasive diagnostic tools like colonoscopy. Actually only a few proteins have been described as biomarkers in CRC (carcinoembryonic antigen (CEA), CA19.9, and CA125 (1–3)), although none of them is recommended for clinical screening (4). Proteomics analysis is actively used for the identification of new biomarkers. In previous studies, the use of two-dimensional DIGE and antibody microarrays allowed the identification of differentially expressed proteins in CRC tissue, including isoforms and post-translational modifications responsible for modifications in signaling pathways (5–8). Both approaches resulted in the identification of a collection of potential tumoral tissue biomarkers that is currently being investigated.However, the implementation of simpler, non-invasive methods for the early detection of CRC should be based on the identification of proteins or antibodies in serum or plasma (9–13). There is ample evidence of the existence of an immune response to cancer in humans as demonstrated by the presence of autoantibodies in cancer sera. Self-proteins (autoantigens) altered before or during tumor formation can elicit an immune response (13–19). These tumor-specific autoantibodies can be detected at early cancer stages and prior to cancer diagnosis revealing a great potential as biomarkers (14, 15, 20). Tumor proteins can be affected by specific point mutations, misfolding, overexpression, aberrant glycosylation, truncation, or aberrant degradation (e.g. p53, HER2, NY-ESO1, or MUC1 (16, 21–25)). In fact, a number of tumor-associated autoantigens (TAAs) were identified previously in different studies involving autoantibody screening in CRC (26–28).Several approaches have been used to identify TAAs in cancer, including natural protein arrays prepared with fractions obtained from two-dimensional LC separations of tumoral samples (29, 30) or protein extracts from cancer cells or tissue (9, 31) probed by Western blot with patient sera, cancer tissue peptide libraries expressed as cDNA expression libraries for serological screening (serological analysis of recombinant cDNA expression libraries (SEREX)) (22, 32), or peptides expressed on the surface of phages in combination with microarrays (17, 18, 33, 34). However, these approaches suffer from several drawbacks. In some cases TAAs have to be isolated and identified from the reactive protein lysate by LC-MS techniques, or in the phage display approach, the reactive TAA could be a mimotope without a corresponding linear amino acid sequence. Moreover, cDNA libraries might not be representative of the protein expression levels in tumors as there is a poor correspondence between mRNA and protein levels.Protein arrays provide a novel platform for the identification of both autoantibodies and their respective TAAs for diagnostic purposes in cancer serum patients. They present some advantages. Proteins printed on the microarray are known “a priori,” avoiding the need for later identifications and the discovery of mimotopes. There is no bias in protein selection as the proteins are printed at a similar concentration. This should result in a higher sensitivity for biomarker identification (13, 35, 36).In this study, we used commercially available high density protein microarrays for the identification of autoantibody signatures and tumor-associated antigens in colorectal cancer. We screened 20 CRC patient and control sera with protein microarrays containing 8000 human proteins to identify the CRC-associated autoantibody repertoire and the corresponding TAAs. Autoantibody profiles that discriminated the different types of CRC metastasis were identified. Moreover a set of TAAs showing increased or decreased expression in cancer cell lines and paired tumoral tissues was found. Finally an ELISA was set up to test the ability of the most immunoreactive proteins to detect colorectal adenocarcinoma. On the basis of the antibody response, combinations of three antigens, PIM1, MAPKAPK3, and ACVR2B, showed a great potential for diagnosis.     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.