Proc. Natl. Acad. Sci. USA Vol. 92, pp. 1213-1217, February 1995 Biochemistry Identification of galectin-3 as a factor in pre-mRNA splicing (CBP35/lectin/RNA processing) SUE F. DAGHER*, JOHN L. WANGt, AND RONALD J. PATFrERSONt§ *Genetics Program and Departments of tBiochemistry and tMicrobiology, Michigan State University, East Lansing, MI 48824 Communicated by Robert L. Hill, Duke University Medical Center, Durham, NC, October 26, 1994 ABSTRACT Galectin-3 (Mr 35,000) is a galactose/lac- and a nonphosphorylated (pl 8.7) form (12). The phosphor- tose-specific lectin found in association with ribonucleoprotein ylated species is found in both the nucleus and cytoplasm, complexes in many animal cells. Cell-free-splicing assays have whereas the nonphosphorylated form localizes exclusively in been carried out to study the requirement for galectin-3 in RNA the nucleus. processing by HeLa cell nuclear extracts by using 32P-labeled Nuclear extracts (NEs) prepared from HeLa cells and MINX as the pre-mRNA substrate. Addition of saccharides that capable of carrying out pre-mRNA splicing contain galectin-3. bind galectin-3 with high affinity inhibited product formation in On the basis of preliminary experiments that showed that the splicing assay, while addition of carbohydrates that do not saccharide ligands with high affinity for galectin-3 can perturb bind to the lectin did not inhibit product formation. Nuclear the splicing reaction, we sought conditions to deplete galec- extracts depleted ofgalectin-3 by affinity adsorption on a lactose- tin-3 from NEs. We now report that extracts depleted of agarose column were deficient in splicing activity. Extracts galectin-3 do not support spliceosome formation or pre- subjected to parallel adsorption on control celiobiose-agarose mRNA splicing. The galectin-3-depleted extracts, however, retained splicing activity. The activity of the galectin-3-depleted regain splicing activity upon reconstitution with the recombi- extract could be reconstituted by the addition ofpurified recom- nant lectin purified from an Escherichia coli expression system. binant galectin-3, whereas the addition of other lectins, either with a similar saccharide binding specificity (soybean aggluti- nin) or with a different specificity (wheat germ agglutinin), did MATERIALS AND METHODS not restore splicing activity. The formation ofsplicing complexes Preparation of NEs and Their Depletion and Reconstitu- was also sensitive to galectin-3 depletion and reconstitution. tion. NEs were prepared from HeLa S3 suspension cultures in Together, these results define a requirement for galectin-3 in buffer D [20 mM Hepes, pH 7.9/20% (vol/vol) glycerol/0.1 M pre-mRNA splicing and identify it as a splicing factor. KCl/0.2 mM EDTA/0.5 mM phenylmethylsulfonyl fluoride (PMSF)/0.5 mM dithiothreitol] as described (13). Typically, Galectin-3 (1) is the name for the galactose(Gal)/lactose NEs with protein concentrations of approximately 18-22 (Lac)-specific lectin previously known under a number of mg/ml were prepared and stored as frozen aliquots at -80°C. different designations, including carbohydrate binding protein Protein concentrations were determined by the Bradford assay 35 (CBP35) (2), Mac-2 (3), IgE-binding protein (4), CBP30 (ref. 14; Bio-Rad). (5), L-29 (6), and L-34 (7). In this communication, we will use NEs were depleted of CBPs by binding them to a-Lac the designation galectin-3 when we refer to the gene or protein insolubilized on 6% beaded agarose (LAC-A), purchased from in general, assuming that studies carried out on the gene or Sigma. Agarose derivatized with cellobiose (CELLO-A) was protein under any one of the above names is applicable to all used as a control for NE depletion. A volume of 200-250 p,l of them. There are instances, however, in which the specific of packed agarose was washed with 20 vol of wash buffer (20 molecule used by one laboratory is slightly (but significantly) mM Hepes, pH 7.9/0.5 M NaCl) in disposable columns different from the corresponding molecule of another labo- (Pierce). NEs were preincubated in buffer 1 (buffer 1 = 60% ratory-e.g., the cDNAs reported for murine CBP35, Mac-2, buffer D) containing 2.5 mM MgCl2, 1 mM ATP, and 5 mM and L-34 are of different lengths. In this case, we will use the creatine phosphate in a volume of 50 ,ul for 30 min at 30°C. old designation to highlight the specific source of the molecule. NaCl was then added to a final concentration of 0.5 M. The galectin family of animal lectins is distinguished by the Preincubated NEs were then added to the washed saccharide- Gal/Lac specificity of its carbohydrate recognition domain agarose and incubated for 30 min at 4°C with rotation. The (CRD), with highly conserved residues between members of unbound fraction was removed from the column. The matrices the family (galectin-1, -2, -3, and -4) and between the homologs were next washed with 50 ,ul ofbuffer 1 containing 0.5 M NaCl, found in various species for any given single member (for and this wash was added to the unbound fraction. The matrices reviews see refs. 8 and 9). The polypeptide of galectin-3 is were washed with 10 vol ofwash buffer, and the bound material delineated into two domains (2, 8): an N-terminal half that is was eluted by boiling the samples in 200 ,ul of Laemmli sample proline and glycine rich, with limited similarity to proteins of buffer (15). Aliquots (20 p,l) of nondepleted NE and the the heterogeneous nuclear ribonucleoprotein (RNP) com- unbound fractions of the plexes, and a C-terminal half that is similar to the CRD of saccharide-adsorption procedure other members of the galectin family. The majority of galec- were dialyzed in a microdialyzer (Pierce) for 40 min against tin-3 is found in the cytoplasm and nucleus of mouse 3T3 buffer 1. fibroblasts in the form of RNP complexes (10, 11). For In reconstitution experiments, recombinant CBP35 example, treatment of permeabilized cells with ribonuclease A (rCBP35) (16) or other lectins (EY Laboratories) were added released the lectin from the nucleus with concomitant loss of to the NE or unbound fractions prior to dialysis. The dialyzed immunofluorescent staining. Two isoelectric variants of ga- lectin-3 have been identified: a phosphorylated (pl 8.2) form Abbreviations: CRD, carbohydrate recognition domain; RNP, ribo- nucleoprotein; NE, nuclear extract; LAC-A and CELLO-A, agarose derivatized with lactose or cellobiose, respectively; TDG, thiodigalac- The publication costs of this article were defrayed in part by page charge toside; CBP, carbohydrate binding protein; Lac, lactose; rCBP35, payment. This article must therefore be hereby marked "advertisement" in recombinant CBP35. accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 1213 Downloaded by guest on September 23, 2021 1214 Biochemistry: Dagher et al. Proc. Natt Acad ScL USA 92 (1995) fractions were then assayed for splicing activity or splicing RESULTS complex formation. Splicing Assay. The plasmid used to transcribe the MINX Effect of Saccharides on Pre-mRNA Splicing. The cell-free pre-mRNA substrate (17) was a kind gift of Susan Berget assay for the splicing of pre-mRNA by HeLa cell NEs was (Baylor College of Medicine, Houston). The MINX pre- optimized for the MINX substrate. In a typical assay (final mRNA was labeled with [32P]GTP and the monomethyl cap protein concentration 10 mg/ml), approximately 20-30% of was added during SP6 polymerase (GIBCO) transcription the input substrate was converted to product (Fig. 1, lane 1). (17). This conversion showed a stringent requirement for ATP (Fig. Splicing reaction mixtures (10 ,ul) contained NE (3 ,ul) or 1, lane 2). When the NE was preincubated with various dialyzed unbound fraction or dialyzed nondepleted NE (8 Al), saccharides (75 mM) prior to the addition of the pre-mRNA 2.5 mM MgCl2, 1.5 mM ATP, 20 mM creatine phosphate, 0.5 substrate, inhibition of product formation was observed for mM dithiothreitol, and 20 units of RNasin (Promega). The Lac (Fig. 1, lane 8), TDG (Fig. 1, lane 4), and melibiose (data final protein concentration of the dialyzed extracts was 7-12 not shown). Other mono- and disaccharides that were tested, mg/ml. For reactions lacking ATP, both ATP and creatine as well as the noncarbohydrate inositol, failed to show inhibi- phosphate were omitted. In experiments testing the effect of tion of product formation. exogenously added carbohydrates, the NEs were preincubated The effect of the inhibitory saccharides was concentration with saccharides at 30°C for 30 min prior to the addition of dependent; half-maximal inhibition was observed at <50 mM 32P-labeled pre-mRNA. for TDG and Lac (Fig. 1, lanes 3-6 and lanes 7-10, respec- Splicing reactions were carried out at 30°C for 45 min. Each tively). Both of these saccharides bind to galectin-3 (20) with splicing sample was diluted to 100 Al with 125 mM Tris, pH approximately the same hierarchy of affinities as that seen in 6.8/1 mM EDTA/0.3 M sodium acetate. Proteinase K was the inhibition of splicing. The disaccharide cellobiose (Fig. 1, added to a final concentration of 2 mg/ml, and the sample was lanes 15-18) and the monosaccharide Gal (Fig. 1, lanes 11-14) digested for 1 h at 37°C. RNA was extracted with 200 ,lI of did not show an effect on the cell-free-splicing assay. Although phenol/chloroform/isoamyl alcohol [50:50:1 (vol/vol)] fol- Gal is a saccharide ligand for the galectin family of lectins, its lowed by 200 ,l of chloroform. RNAs were precipitated with affinity for binding galectin-3 is about two orders ofmagnitude 300 ,ul of ethanol in a dry ice/ethanol bath.