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SITY LIBRARIES Millilllllllllllllllllllll”ll“ lllllllll This is to certify that the dissertation entitled The Characterization of Nuclear Lectins as Novel Splicing Factors presented by Sue Fay Dagher has been accepted towards fulfillment of the requirements for Ph . D . degree in Genetics flimflw Majop pffessor Date 7'12"?“ MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY Mlchlgan State UnIverslty w— PLACE II RETURN BOXto manual. mum your record. TO AVOID FINES Mum on or More data duo. DATE DUE DATE DUE DATE DUE THE CHARACTERIZATION OF NUCLEAR LECTINS As NOVEL SPLICING FACTORS By SUE FAY DAGHER A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Genetics Program 1994 ABSTRACT THE CHARACTERIZATION OF NUCLEAR LECTINS AS NOVEL SPLICING FACTORS By SUE FAY DAGHER Galactose/lactose specific lectins (galectins) have been identified in the nuclear compartment of a number of mammalian cells; Immunofluorescent studies have shown that galectin—3 is one such lectin. Previous results have shown galectin-3 is a component of ribonucleoprotein complexes, which has lead to testing for its possible role in pre-mRNA splicing. The addition of various saccharides to nuclear extracts was used to evaluate whether galectin-3 specific saccharides perturbed pre-mRNA splicing. Galactose containing saccharides as well as neoglycoconjugates with a high affinity for galectin-3 perturbed in vitro pre-mRNA splicing reactions. Mono- and disaccharides without affinity for galectin-3 had little effect on splicing activity. To specifically implicate galectin-3 to the saccharide inhibition results, nuclear extracts were immunodepleted of galectin-3. Although greater than 95% of galectin-3 is removed, splicing activity is only slightly diminished compared to a control depleted extract. However, when lactose is added to the galectin-depleted extracts, splicing is still inhibited. These results suggest another lactose binding protein(s) may be influenced by the addition of exogenous saccharides. To remove all lactose binding proteins from nuclear extracts, depletions were performed using saccharide-immobilized affinity matrices. When tested for splicing activity, lactose depleted extracts were unable to process pre-mRNA while control cellobiose depleted extracts retained full splicing activity. Most importantly, splicing activity is restored to lactose depleted extracts by the inclusion of recombinant galectin-3 (rCBP35) or recombinant galectin-l (rL-14). We suggest that several lactose binding proteins exist in splicing extracts which are functionally redundant. Removal of one lactose binding protein permits the others to act in pre-mRNA processing, while removal of all of them abolishes splicing activity. DEDICATION TO MY PARENTS Who always stood by me, always bad faith in me and understood my love for science. iv ACKNOWLEDGMENTS First of all I would like to say that words alone cannot describe the patience shown and guidance given by Ron Patterson, my mentor, throughout my training. Ron was always ready to listen to my ideas and advise me when necessary. Over the years we worked as a team while performing experiments and designing experimental strategies to tackle new questions. All of this and much more has made science and its study the most exciting and fulfilling field to be involved in. I want to thank my committee members Sue Conrad, Donna Koslowsky, Natasha Raihkel and Richard Schwartz for their guidance and helpful advise. It has also been a privilege to collaborate with John Wang and the members of his laboratory who provided advise and materials that enhanced my research. Finally, I want to thank two good friends Trifon Adamidis and Georg Hausner for tolerating my continued tardiness when I was delayed with an experiment. TABLE OF CONTENTS PAGE LIST OF TABLES ............................................. ix LIST OF FIGURES ............................................. x LIST OF ABBREVIATIONS ...................................... xii CHAPTER I: LITERATURE REVIEW ............................... 1 INTRODUCTION TO LECTINS ............................. 2 Lectin overview ...................................... 2 A historical prospective of the galectin-3 S-lac proteins ............................................ 3 Putative functions for galectin-3 surface proteins ............. 6 Nuclear lectins ...................................... 7 Nuclear glycoproteins ................................. 8 Carbohydrate Binding Protein 35: (CBP35) ................. 9 REVIEW OF pre-mRNA SPLICING .......................... 12 Mechanism of pre-mRNA Splicing ....................... 12 Non-snRNP protein splicing factors ...................... 18 HnRNP proteins .................................... 20 SAPS ............................................. 22 CONCLUSION: Nuclear galectins and pre-mRNA splicing: is there a connection? ........................ 24 BIBLIOGRAPHY ........................................ 25 CHAPTER II: IDENTIFICATION OF GALECI‘IN-3 AS A REQUIRED FACTOR IN pre-mRNA SPLICING .................... 33 FOOTNOTES ........................................... 34 ABSTRACT ............................................ 35 INTRODUCTION ........................................ 36 MATERIALS AND METHODS ............................. 37 Preparation of Nuclear Extracts and Their Depletion and Reconstitution .................................. 37 Splicing Assay ...................................... 39 Gel Mobility Shift Assay For Splicing Complex Formation ......................................... 40 Western Blot Analysis ................................ 40 RESULTS .............................................. 41 Effect of Saccharides on pre-mRNA Splicing ............... 41 Splicing Activity of Galectin-Depleted Extracts ............. 44 Reconstitution of Splicing in Galectin-B-Depleted Extracts ........................................... 47 Analysis of Spliceosome Complex Formation in Original, Depleted, and Reconstituted Extracts ..................... 52 DISCUSSION ........................................... 60 REFERENCES .......................................... 65 CHAPTER III: Nuclear Galectins: A Family of Functionally Redundant Splicing Factors INTRODUCTION ........................................ 68 MATERIALS AND METHODS ............................. 70 Immunodepletion and Reconstitution ..................... 70 RESULTS ......................................... 71 Identification of galectins in splicing extracts ............... 71 Galectin-immunodepletion from splicing extracts ............ 76 Double galectin immunodepletion ....................... 81 Galectin reconstitution of splicing activity ................. 81 DISCUSSION ........................................... 89 REFERENCES .......................................... 91 vii ADDENDUM CHAPTER IV: PHOSPHORYLATION AND UV CROSS-LINKING ........ 93 Phosphorylation .......................................... 94 INTRODUCTION ........................................ 94 MATERIALS AND METHODS ............................. 95 In Vztro Phosphorylation .............................. 95 RESULTS .............................................. 96 DISCUSSION .......................................... 101 Photochemical Cross-Linking of Galectin-3 to pre-mRNA During pre-mRNA Splicing ....................... 104 INTRODUCTION ....................................... 104 MATERIALS AND METHODS ............................ 104 Pre-mRNA synthesis and in vitro splicing conditions ......... 104 UV cross-linking ................................... 104 Preparation and analysis of UV cross-linked proteins ........ 105 RESULTS ............................................ 105 DISCUSSION .......................................... 111 REFERENCES ......................................... 112 CHAPTER V: CONCLUSION ................................... 114 Conclusions and Future Experimentation ...................... 115 viii LIST OF TABLES PAGE CHAPTER I Table 1 Mammalian snRNP proteins associated with snRNAs involved in pre-mRNA splicing .................................. 16 Table 2 Mammalian splicing factors ............................ 23 ix LIST OF FIGURES PAGE CHAPTER I Figure 1 The Two Transesterification Steps in pre-mRNA Splicing ...... 15 Figure 2 The Pathway of pre-mRNA Splicing in Mammalian Cells ...... 17 CHAPTER II Figure 1 The effect of saccharides on pre-mRNA splicing ............ 43 Figure 2 Concentration dependence of the TDG and Lac on pre-mRNA splicing ........................................... 46 Figure 3A Comparision of the levels of galectin-3 in NE and in the U8 and B fractions when NE were subjected to adsorption on LAC-A and CELLO-A ............................... 49 Figure 3B Comparision of splicing activity of NE and the UB fraction of LAC-A and CELLO-A affinity adsorptions .............. 51 Figure 4 The effect of rCBP35 and soybean agglutinin on the splicing activity of the UB fraction of LAC-A ..................... 54 Figure 5A Concentration dependence of the reconstitution of splicing activity in the UB fraction of LAC-A by rCBP35 ............ 56 Figure 5B Formation of spliceosomal complexes by the UB fraction of the LAC-A adsorption in the absence and presence of rCBP35 . . . . 59 CHAPTER III Figure 1 Detection of galectin-1 and galectin-3 in LAC-A bound NE . . . . 73 Figure 2 Localization of galectin-1 and galectin-3 by immunofluorescence staining of NIH 3T3 fibroblasts ......... 74 Figure 3A The effect of galectin-3 on pre-mRNA splicing .............. 78 X Figure 38 Comparison of the levels of galectin-3 in NE and the UB and B fractions of NE after immunodepletion with aTR, aM2 and aSm ................................. 80 Figure 4A Comparison of splicing activity of NE immunodepleted of galectin-1 and/ or galectin-3 ............................ 83 Figure 4B Comparision of the levels of galectins in NE and in the U8 and B fractions subjected to immunodepletion by aM2 and aLl4 separately and together ....................... 85 Figure 5 The effect of rCBP35 and rL14 on the splicing activity of the UB fraction of LAC-A ................................ 88 CHAPTER IV Figure 1A Proteins phosporylated during in vitro splicing .............. 98 Figure 13 Immunoblot analysis of rCBP35 and galectin-3 following in vitro phosphorylation of a splicing reaction ............... 100 Figure 1C Immunoblot analysis of C1 and C2 proteins following in vitro phosphorylation of a splicing reaction .............. 100 Figure 2A UV cross-linked proteins following in vitm splicing .......... 108 Figure 2B Immunoblot analysis of galectin-3 following UV cross-linking of a splicing reaction ............................... 110 Figure 2C Immunoblot analysis of C1 and C2 proteins following UV cross-linking of a splicing reaction ...................... 112 ATP B CBP35 Cello CELLO-A CM-sepharose CP CRD DME DNase Gal Glu HMG hnRNP LAC-A NE LIST OF ABBREVIATIONS adenosine triphosphate bound carbohydrate binding protein 35 cellobiose beaded agarose derivitized with cellobiose carboxymethyl-sepharose creatine phosphate carbohydrate recognition domain Dulbecco’s modified Eagle’s medium deoxyribonuclease Fluorescein isothiocyanate galactose glucose high mobility group protein heterogeneous nuclear ribonucleoprotein complex lactose beaded agarose derivitized with lactose nuclear extract xii PAGE PBS PBS-T PMSF RNase SBA SDS snRNP UB WGA polyacrylamide gel electrophoresis phosphate buffered saline phosphate buffered saline + Tween 20 phenylmethylsulfonyl fluoride ribonuclease soybean agglutinin sodium dodecyl sulfate small nuclear ribonucleoprotein particle thiodigalactoside unbound wheat germ agglutinin xiii CHAPTER I LITERATURE REVIEW 2 INTRODUCTION TO LECTINS Lectin overview Lectins are non-enzymatic non-immunoglobin proteins that bind carbohydrates. Lectins also agglutinate cells and precipitate polysaccharides and glycoproteins. These latter properties are due to the polyvalent nature of lectins; each lectin molecule has at least two carbohydrate binding sites to allowing crosslinking between cells or between glycoproteins (Sharon and Lis, 1989). Lectins have been found in plants, mammals, microorganisms and viruses. They are structurally diverse and in general are found as oligomeric proteins composed of subunits, usually with one carbohydrate recognition domain (CRD) per subunit. They vary, however, in amino acid composition, molecular weight, metal requirement, and three dimensional structure. Despite this variability, they can be grouped into families of structurally homologous proteins. For more detailed information refer to a comprehensive review by Liener et a1. (1986). The purpose of this review is to give an overview of the properties of mammalian lactose binding lectins (galectins, see below) emphasizing CBP35 (carbohydrate binding protein Mr=35,000). A variety of lactose binding proteins have been classified as soluble lactose binding lectins (S-Lac) (Drickamer, 1988). S-lac lectins are a family of vertebrate proteins which share homologous lactose CRD, do not require divalent cations for carbohydrate binding and are soluble without detergents (Drickamer, 1988; Leffler et al., 1989). Although grouped together partially on the basis of their ability to bind lactose, subtle differences in carbohydrate binding 3 specificities do exist (Leffler and Barondes, 1986). Recently a more comprehensive nomenclature for S-Lac lectins has been established. S-Lac lectins can be grouped into one of four galectin families (Barondes et al., 1994). For example, the galectin- 3 farme includes CBP35, Mac-2, IgEBP, CBP-30, RL-29, L-29, L—31, L-34, and LBL (Barondes et al., 1994). The galectin-1 family includes b14-1, L-14, RL-14.5, galaptin, MGBP, GBP, BHL, CHA, HBP, HPL, HLBP 14, and rIML-l (Barondes et al., 1994). A number of investigators have attached putative functions to many of the galectins (Harrison, 1991; Hamann et al., 1986, Frigeri and Liu, 1992, Mercurio and Shaw 1991, Cooper et al., 1991) which are presented later in more detail. A historical prospective of the galectin-3 S-lac proteins Over the past 14 years members of galectin-3 proteins have been identified in a number of mammalian cells. Although independently characterized and named based on discovery, all possess 80-90% homology to the originally described murine Mac-2 protein (Ho and Springer, 1982) and contain the galectin fi-galactose binding motif (Drickamer et al., 1988). The chronological list includes: rat lung lectin (RL- 29; Cerra et al., 1985), mouse tumor lectin (mL-34; Raz et al., 1986), human lung lectin (HL-29; Sparrow et al., 1987), rat immunoglobulin E-binding protein (IgE BP; Albrandt et al., 1987), carbohydrate binding protein (CBP35; Moutsatsos et al., 1987), mouse macrophage antigen (Mac-2; Cherayil et al., 1989), rat, mouse and human 29 kDa galactose binding lectin (L-29; Oda et al., 1991), human breast carcinoma galactoside binding lectin (hL-29; Oda et al., 1991) and human tumor-associated 31 kDa galactoside-binding lectin (hL-31; Raz et al., 1991). 4 Mac-2 was first observed as an inflammation responsive protein on the surface of macrophages which increases during inflammation suggesting an important role in the inflammatory response (Ho and Springer, 1982). In addition Mac-2 is the major nonintegrin laminin-binding protein synthesized by murine inflammatory macrophages, pointing to a potential role for Mac-2 in macrophage-extracellular interactions (Woo et al., 1990). The human homologue of Mac-2 (hMac—2) has been cloned and shown to have a highly conserved primary structure when compared to murine Mac-2 with 85% of the amino acids being similar (Cherayil et al., 1989, 1990). The in vitro synthesized hMac-2 has the galactose specific binding property of other [’29 lectins, and binds to purified laminin (Cherayil et al., 1989). A galactose binding protein which localizes on the cell surface, in the cytoplasm and the nucleus of 3T3 cell mouse fibroblasts has been identified and named carbohydrate binding protein 35 (CBP35) (Moutsatsos et al., 1987). Immunofluorescent analysis of 3T3 cells indicated CBP35 is a component of ribonucleoprotein complexes (J ia and Wang, 1988). The same protein was identified and named L-34 and suggested to be a tumor cell surface lectin that enhanced tumor metastasis by promoting the formation of multicellular emboli (Raz et al., 1989). An IgE binding protein found in the cytosol of rat cells (Liu et al., 1985) also has extensive homology to the galectin-3 lectins found thus far. CBP35 (Laing et al., 1989) and Mac-2 (Cherayil et al., 1989) also have the ability to bind murine IgE and this binding can be inhibited by galactose, which raises the possibility that galectin-3 species may be involved in the biosynthetic or functional regulation of IgE (Liu et al., 1985). The classic definition of a lectin is a class of proteins that bind carbohydrates to their CRD. However, we 5 should not rule out the possibility that glycoproteins and nonglycoproteins could interact with lectins at regions other than the CRD and this interaction may be altered by carbohydrates specific for the lectin. Such a situation may exist between IgE and IgE binding protein (Liu et al., 1985) since it is not known whether their interaction is through a galactose containing side chain or an IgE binding site on IgE binding protein (Cherayil et al., 1989). The galectin-3 proteins cloned and sequenced so far possess two-domains. The carboxyl-terminal carbohydrate binding domain is particularly conserved in which long, uninterrupted stretches of amino acid identity can be found. The amino- terminal domain contains PGAYPG repeats whose number varies between Mac-2, hMac-2, L-34 and CBP35. The function of these amino-terminal repeats is unclear, however their evolutionarily conserved nature suggests an important role or function for the lectin. The following common characteristics have been summarized for the galectin-3 family of galectins: i) molecular weights between 26,200 and 30,300, ii) composed of two structural domains; the amino-terminal domain contains a number of proline glycine-rich tandem repeats, and the carboxyl-terminal domain contains a 6-galactoside CRD, iii) expression by various cell types - both intracellular and as surface molecules, iv) highly conserved amino acid sequence among different species, v) absence of known transmembrane domain and signal sequence. Putative functions for galectin-3 surface proteins Although the galectin-3 proteins have been characterized for only 14 years the amount of information gathered has been bountiful as well as diverse. 6 Immunohistochemical, cell surface labeling, and ligand binding studies have shown galectin-3 proteins to localize in the cytosol, nucleus, and on the cell surface. As cell surface moieties they are assumed to function by interacting with complementary glycoconjugates (Frigeri and Liu, 1992; Gritzmacher et al., 1988; Moutsatsos et al., 1987; Raz et al., 1984; Weis et al., 1991). Woo et a1. (1990) have shown Mac-2 may have an important role in the inflammatory process. Mac-2 is the major non-integrin laminin-binding protein expressed by murine peritoneal macrophages exposed to thioglycollate. Consequently, Mac-2 may aid in facilitating adhesion to the basement membrane laminin. A number of studies have provided further information on interactions between lectins and laminin and their functional significance (Bouzon et al., 1990; Cooper et al., 1991; Dean et al., 1990; Mercurio and Shaw, 1991; Sato and Hughes, 1992; Zhou and Cummings, 1990) Briefly, these studies suggest that lectin-laminin interactions may actually impair cell-cell adhesion by blocking laminin interaction with a cell surface receptor such as an integrin near an oligosaccharide. The metastatic state of melanoma and fibrosarcoma cells appears to correlate with the level of expression of L-34 (Raz et al., 1986; 1989) and metastasis of these cells could be inhibited by the addition of monoclonal antibody to L-34 (Meromsky et al., 1986). Cells transfected with cloned cDNA provided further evidence for the role of this lectin in tumor metastasis (Raz et al., 1991). These findings along with those obtained by Woo et al. (1990) demonstrate that Mac-2 may play a role in crossing the basement membrane via galactose containing sugar side chains of the basement laminin. Cherayil et al. (1990) suggest using the monoclonal antibody M3 /38 which cross reacts with both murine and human Mac-2 as a tool to investigate the 7 involvement of human Mac-2 in human diseases. Nuclear lectins The existence of nuclear lectins has been reported in mammalian cells (Bourgeois et al., 1987; Facy at el., 1990; Laing et al., 1988; Moutsatsos et al., 1986, 1987; Olins et al., 1988; Seve et al., 1985, 1986), reptilian cells (Hubert et al., 1985) and in the macronucleus of protozoans (Olins et al., 1988). Strong evidence indicates nuclear lectins may be involved in cell proliferation (Hubert et al., 1989; Wang et al., 1991). Quantitative changes in nuclear and cytoplasmic lectins have been reported in conjunction with the proliferative state of the cell (Bourgeois et al., 1987; Moutsatsos et al., 1986, 1987; Seve et al., 1986) and to the cell cycle (Moutsatsos et al., 1986; Facy et al., 1990). Lower amounts of nuclear lectins were found in differentiated cells compared to undifferentiated cells (Facy et al., 1990). Further examination of nuclear lectins has revealed a preference for localization in regions enriched for ribonucleoprotein complexes (RNP) (Bourgeois et al., 1987; F acy et al., 1990; Hubert et al., 1985; Hubert et a1 1989; Laing et al., 1988; Jia et al., 1988; Seve et al., 1985; Seve et al., 1986) and at the site of DNA replication in the macronucleus of Euplotes ewystomus (Olins et al., 1988). In higher eucaryotes these could be sites of transcriptional events, posttranscriptional modifications and regions RNPs are readied for export to the cytoplasm. Analysis of binding of D-glucose and N-acetylglucosamine containing neoglycoproteins to nuclei from the the human tumor cell line HL-60 has been shown (Facy et al., 1990). A glucose binding protein (CBP67) isolated from rat liver 8 nuclei is present in nuclear RNP but absent in polysomal RNP indicating a possible role in nucleocytoplasmic export of mRNA (Schroder et al.,1992). It is possible that nuclear lectins mediate posttranscriptional processing and perhaps export of mRNAs specific for synthesis of proteins required for cell proliferation and DNA replication (Facy et al., 1990). In support of this idea, a set of hnRNPs was found to modulate cell proliferation during differentiation of 3T3 cells and normal keratinocyes in this manner (Minoo et al., 1989). Nuclear glycoproteins Theoretically, one would expect nuclear glycoproteins to serve as ligands for nuclear lectins. The existence of nuclear glycoproteins is well established (for reviews refer to Hart et al., 1989; Wang et al., 1991). It has been postulated that specific nuclear functions may be modulated by interactions between nuclear lectins and nuclear glycoproteins (Hubert et al., 1989) A series of nuclear glycoproteins have been identified and may serve as potential ligands [for example, the O-linked glyc0proteins poly(A) polymerase (Kurl et al., 1988), several RNA polymerase II transcription factors (Jackson and Tjian, 1989; Lichtsteiner and Schibler, 1989) as well as N-linked glycoproteins; high mobility group proteins HMG14 and HMGl7 (Reeves and Chang, 1983)]. In addition, an hnRNP (p43, Mr 43,000) has been characterized as a glycoprotein, bearing N-aceylglucosamine oligosaccharide residues (Soulard et al., 1991). Glycoproteins have been found on the cytoplasmic and nucleoplasmic surfaces of nuclear pore complexes (Davis and Blobel, 1986; Hanover et al., 1987; Holt et al., 9 1987) and associated with the nuclear matrix (Reeves and Chang, 1983). Nuclear protein import is blocked by the lectin wheat germ agglutinin which recognizes residues within the pore complex bearing O-linked N-acetylglucosamine (Finlay et al., 1987; Yoneda et al., 1987). On the other hand wheat germ agglutinin does not inhibit RNA export from isolated nuclei (Schroder et al., 1986). Nuclear export of mRNA and nuclear envelope nucleoside triphosphatase activity is inhibited by the lectin (Gerardia Savagia) specific for D-mannose bearing pore complex glycoproteins (Kljajic et al., 1987). Although the list of nuclear glycoproteins continues to grow rapidly, no one has shown that any of these glycoproteins act as ligands for nuclear lectins. Carbohydrate Binding Protein 35 (CBP35): galectin-3 About 10 years ago NIH 3T3 cell extracts were fractionated over an asialofetuin column (Roff and Wang, 1983). Three bound proteins could be eluted from the column with lactose (galactose, 31-4 glucose). Two of the proteins had molecular weights of 14 kDa (galectin-1) and 16 kDa. The third had a molecular weight of 35 kDa and was designated CBP35 (now galactin-3). The research that followed focused on this lectin. As previously mentioned galectin-3 proteins possess N-terminal domains not present in galectin-1 proteins. The carboxyl-domain of galectin—3 contains sequences homologous to other B-galactoside binding proteins, while the amino-terminal domain possesses a distinct repeated pattern of PGAYPG (Jia and Wang, 1988). Galectin-3, originally identified as an extracellular component known as Mac-2 (Ho and Springer, 1982), has been found in the cytoplasm, nucleus and on 10 the surface of numerous mammalian cells (Moutsatsos et al., 1986). Human Mac-2 has been detected in fibroblast cell lines and various epithelial cell lines, however it has not been detected in the lymphoid cell line BJAB, (Cherayil et al., 1990). Galectin—3 has been detected in all cell lines tested with the exception of the lymphoid cell line D1C1 (s. D., unpublished observations). The pI of galectin-3 has been determined both experimentally and by calculation from the deduced 264 amino acid sequence (Cowles et al., 1990). When extracts of NIH 3T3 cells were subjected to NEPHGE‘ and immunoblotted, two spots corresponding to pI 8.7 and 8.2 were observed (Cowles et al., 1990). The pI 8.2 form is the result of a post-translational modification of the pI 8.7 by the addition of a single phosphate group. Recently the L-29 galectin-3 has been shown to be phosphorylated predominatly (90%) at Ser6 and to a lesser extent (10%) at Ser12 (Huflejt et al., 1993). These two isoelectic species were found to be differentially expressed in the nucleus and cytoplasm of NIH 3T3 cells during quiescence, serum stimulation, and development (Moutsatsos et al., 1987; Agrwal et al., 1989). The cytoplasm contains predominantly the phosphorylated form of galectin-3. Both species are found in the nucleus. In quiescent cultures of NIH 3T3 cells galectin-3 is mainly found at low levels in the cytoplasm and only the phosphorylated form can be detected in the nucleus. In proliferating cultures the amount of phosphorylated galectin-3 increases both in the nucleus and cyt0plasm with the most dramatic increase seen in the nuclear non-phosphorylated species (Cowles et al., 1990). Immunofluorescent studies have shown that various enzymatic and biochemical treatments of cells such as high salt extraction and DNAase-treatment did not result 11 in the removal of nuclear galectin-3. However, RN Aase-treatment and salt extraction resulted in quantitative removal of galectin-3 from nuclei (Laing and Wang, 1988). Lactose-affinity chromatograpy of nuclear extracts selected galectin-3 and several other proteins whose molecular weights matched those of hnRNP proteins (Wang et al., 1992; Laing and Wang, 1988). The nuclear localization of galectin-3 and possible association with hnRNP proteins has stimulated inquiry into its putative role in pre- mRNA splicing. The first indication that galectin-3 might be involved in the splicing process appeared when Lac and neoglycoconjugates containing Lac were tested for their ability to perturb pre-mRNA splicing. Lac inhibited in vitro pre-mRNA splicing in a concentration dependent manner while saccharides that did not have an affinity for galectin-3 had very little effect on splicing (Wang et al., 1992). More recently a 70 kDa glucose binding lectin (CBP70) was isolated in a complex with galectin-3 from HL60 membrane-depleted nuclei and this association was controlled by the binding of galectin-3 to Lac (Seve et al., 1993). However, we have been unable to coprecipitate CBP70 and galectin-3 using glucose affinity resins (data not shown). In summary, i) galectin-3 can be found extracellularly, in the cytoplasm and in the nucleus, ii) the amount and phosphorylation state of galectin-3 varies according to the proliferative state of the cell culture, iii) nuclear galectin-3 was found associated with RNA as component of ribonucleoprotein complexes, iv) specific disaccharides with a high affinity for galectin-3 inhibited in vitro splicing activity, whereas, mono— and di-saccharides without affinity for galectin-3 had little effect on splicing activity. These preliminary findings are the basis for the continued 12 examination of the involvement of galectin-3 in pre-mRNA splicing and the subject of this thesis. REVIEW OF pro-mRNA SPLICING Mechanism of pre-mRNA splicing Pre-mRNA splicing proceeds through a two step mechanism involving two separate trans-esterification reactions (Figure 1). The first step includes cleavage at the 5’ splice site generating free exon 1 with a 3’-OH terminus and exon 2-intron lariat. The lariat structure results from the formation of a 2’-5’ phosphodiester bond linking the 5’ terminus of the intron to the ribose 2’ OH group of an adenosine residue in the intron creating a "branch point". The second step of the splicing reaction involves cleavage of the 3’ splice site and ligation of exon 1 and exon 2 generating the spliced mRNA and the excised lariat intron (for a recent review, refer to Lamond, 1993). In vitro splicing of pre-mRNA involves the formation of several splicing complexes including spliceosomes by way of ordered assembly of small nuclear ribonucleoprotein particles (snRNPs) (Table 1) and non-snRNP protein splicing factors on the pre-mRNA (Table 2) (for a review, refer to Luhrmann et al., 1990). The snRNPs involved in splicing are referred to as U1, U2, U4 / U6 and U5 snRNPs. The snRNAs Ul-US are transcribed by RNA polymerase II and snRNA U6, by RNA polymerase III. With the exception of U6, newly transcribed monomethyl G-capped U snRNA is transported to the cytoplasm where the cap is modified to a trimethyl 13 G structure and the snRNA assembles with proteins to form the snRNP (for a review, refer to Mattaj, 1988). Many of the snRNAs complex with a set of core proteins, B, B’, D, D’, E, F, and G which serve as antigens for human anti-Sm autoimmune antibodies in many patients with connective tissue disease (Lerner et al., 1979). Several snRNPs also have U specific proteins such as the U1 70 kDa, A and C proteins, and the U2 A’ and B” proteins (Table 1). Figure 2 displays several known steps that take place leading to spliceosome formation. Prior to formation of the first splicing complex newly transcribed pre-mRNA is bound by hnRNP proteins to form the hnRNP complex or H complex. Formation of the H complex does not require functional 5’ or 3’ splice sites thus, excluding its formation as a functional intermediate in spliceosome formation (Konarska and Sharp, 1987). The first of several steps leading to spliceosome formation is the ATP independent interaction of U1 snRNP with both the 5’ and 3’ regions of the intron forming the "commitment complex". Next a pre-spliceosome complex referred to as the "A complex" is formed by the ATP dependent binding of U2 snRNP to the pre-mRNA branch site. Binding of U2 snRNP requires U1 snRNP (Barabino et al., 1990) and three protein factors: SFl, SF3 (Kramer and Utans, 1991) and U2 auxiliary factor (U2AF) (Ruskin et al., 1988; Zamore and Green, 1989). The spliceosome or "B complex" is formed following the addition of a preassembled U4/U6 U5 tri-snRNP particle and at this point the two transesterification reactions take place within the spliceosome complex. Other putative mammalian splicing factors in addition to SF2/ASF are SF 1 (containing U1 and U2 snRNPs), SF3, SF4A and SF4B (Table 2) (Krainer et al., 14 1985). SFl, SF2 and SF4B are required prior to the 5’ splice site cleavage step and SF3 and SF4A are required for the second catalytic step of splicing (Mayeda and Krainer, 1992). Another set of splicing factors named SFl‘, SF2“, SF3“ and SF4“ are required for 5’ splice site cleavage and lariat formation (Table 2) (Kramer et al., 1987; Utans and Kramer, 1990) (The ""' is used to differentiate SF2 and SF3 found by Mayeda and Krainer, 1992). Galectin-3 has not been found associated with snRNPs either by immunoprecipitation with anti-M2 (a monoclonal directed against Mac-2) or with anti-Sm (a polyclonal antibody directed against specific Sm proteins). However Lac affinity chromatography of nuclear extracts co-selected with galectin-3 several proteins whose molecular weights were characteristic of hnRNP proteins (Wang et al., 1992; Laing and Wang, 1988). I have therefore concentrated the remaining review on a set of splicing factors not associated with snRNPs. 15 Figure l The Two Transesterification steps in pre-mRNA Splicing BRANCH POINT 5’ EXON 1 AG pGU A ' Y(n$\lCAG pG EXON 2 3’ ‘x / “‘~--.‘___ ............ OH 2 pro-mRNA 5’splice site cleavage T P1 S E EXON 2-INTRON LARIAT FORMATION Y a: 92 0 Z > G) o 3' 09 5- =.= D i A 2, Y(n5\lCAG pG EXON 2 3 3’ splice site cleavage STEP2 EXON 1-EXON 2 LIGATION ' U9 5' g 5’ EXON1 |b EXON2 3’ P i A—Y CAG 2' (n mRNA INTRON LARIAT 16 Table 1 Mammalian snRNP proteins associated with snRNAs involved in pro-mRNA splicing. The snRNP proteins associated with each snRNA shown to be involved in mammalian pre-mRNA splicing are listed along with protein mass and RNA sizes wherever possible. Core proteins and their respective masses are listed at the end of the table. The data presented is compiled from Reddy, 1986: Bennett et al., 1992 and Lamond, 1993. RNA nt length proteins molecular mass (kDa) U1 165 core 70K 70 A 34 C 22 U2 188 core A’ 31 B” 28.5 U4 143 core US 106 200 116 112 110 40 15 U6 57 core B 28 B’ 29 D 16-18 E 12 F 11 G 9 17 Figure 2 The pre-mRNA splicing pathway in mammalian cells 56¢ij pa“ {radium 5| :1 If Won <3 14' £2 [3' pro-mRNA hnRNP proteins 0 'C 5" £1 lfw {‘r { E2 I 3' HcompIex @ +ATP U1 5' :1 O { 22 I 3' commitment complex IBP @ U2AF non snRNP splicing factors ’ SF1+SF3 A complex Spliceosome lntron 5' l E1 I 52 3' mRNA 18 Non-snRNP protein splicing factors Certain non-snRNP proteins have been identified in mammalian cells which are found to be essential for splicing activity in vitro and/or are stably associated with splicing complexes (Table 2). A comparison of sequences of these factors indicates they contain two classes of motifs found in other splicing factors: an arginine/serine- rich (RS) motif and an RNA recognition motif (RRM) (Table 2) (Lamrn and Lamond, 1993). Certain RS regions of U2AF“s (Zamore et al., 1992) and the splicing factor SF2/ASF (Ge et al., 1991; Krainer et al., 1991) were found to be similar to those of U1 snRNP 70k protein (Mancebo et al., 1990), and the Drosophila splicing regulators transformer; (Boggs et al., 1987), transformer-2 (Amrein et al., 1988), suppressor-o -sable (Voelker et al., 1991) and suppressor-of-apn'cot (Chou et al., 1987). U2AF35 also contains the SR domain, however it lacks the RRM and does not bind directly to RNA (Zhang et al., 1992). The location, length and composition of the SR motif varies among splicing factors. Both SF2/ASP and SC35/PR264 contain the distinctive RRM and RS motif, however with only 31% amino acid sequence identity (Bandziulis et al., 1989; Kenan et al., 1991). SF2/ASF protein is seen to vary in abundance in different rat tissues (Fu et al., 1992) and variable levels of SC35/PR264 mRNAs are expressed in different cell lines and stages of development (Fu and Maniatis, 1992; Vellard et al., 1992). The possible functions of these non-snRNP splicing factors are discussed below. U2AF (U2 snRNP auxilliary factor) is composed of a 65 kDa protein (U2AF‘5) and an associated 35 kDa protein (U2AF35) (Zamore and Green, 1989). So far only the 65 kDa polypeptide is required for in vitro splicing of pre-mRNA (Zamore and 19 Green, 1989). Prior to spliceosome formation U2AF“ binds to the pre-mRNA polypyrimidine tract in an ATP independent fashion and is believed to stablize the ATP dependent binding of U2 snRNP to pre-mRNA during splicesome assembly (Ruskin et al., 1988). U2AF has also recently been shown to play a role in alternative splicing (Zamore et al., 1992). SF2 (splicing factor 2) (Krainer and Maniatis, 1985; Krainer et al., 1990) also known as ASF or ASF-1 (alternative splicing factor) (Ge and Manley, 1990; 1991) is a phosphoprotein with an apparent molecular mass of 27 kDa (Ge et al., 1991; Krainer et al., 1991). SF2/ASF has been shown to be required for the first step in assembly of the first detectable ATP dependent presplicesome complex (A complex) (Krainer et al., 1990; Ge and Manley, 1990). SF2/ASF has been shown to be involved in 5’ splice site selection, activating the proximal 5’ splice site in pre-mRNAs containing two or more 5’ splice sites (Eperon and Krainer, 1993; Ge and Manley, 1990; Harper and Manley, 1992; Krainer et al, 1990). In addition SF2/ASF preferential splice site selection can be counteracted by A1 hnRNP (Mayeda and Krainer, 1992) which is thought to be antagonistic to the binding of U1 snRNP (Buvoli et al., 1992; Eperon et al., 1993). Both SF2/ASF and hnRNP A1 have strand annealing activities which may promote base pairing of snRNAs to the intron at alternative splice sites (Krainer et al., 1990; Kumar et al., 1990; Munroe et al., 1992; Pontius et al., 1990). SC35 (splicesome component of 35 kDa) (Fu and Maniatis, 1990; 1992a; 1992b) also named (PR264) (Vellard et al., 1992) is a phosphoprotein required for the first ATP dependent step in splicesome assembly and appears to be one of the factors 20 that mediate U1 snRNP and U2 snRNP interactions at the 3’ splice site (Fu and Maniatis, 1992a; 1992b). Similar to SF2/ASF, SC35/PR264 favors proximal over distal 5’ splice sites and is counteracted by hnRNP A1 (Fu and Maniatis, 1990; Spector et al., 1991). Unaffected by hnRNP A1 is the ability of both SF2/ASF and SC35/PR264 to influence 3’ splice site selection in pre-mRNA containing competing 3’ splice sites (Fu et al., 1992). SF2/ASF can compliment extracts immunodepleted of SC35/PR264 (Fu et al., 1992) and both SF2/ASF and SC35/PR264 can complement S100 extracts for splicing (Fu et al., 1992; Krainer and Maniatis, 1985). Recently it has been suggested that both SC35 and SF2/ASF play a role in modulating alternative 3’ and 5’ splice site selection, favoring proximal splice site selection at both ends of the intron which may be important for preventing exon skipping in complex pre-mRNAs containing multiple introns (Fu et al., 1992). Finally SF2/ASF and SC35/PR264 appear to be redundant in vitro splicing factors with highly related but distinct amino acid sequences (Fu et al., 1992). HnRNP proteins Several hnRNP proteins also subscribe to a set of non-snRNP protein splicing factors. HnRN P proteins are found on the first detectable splicing complex (Bennett et al., 1992a) and can be detected in purified spliceosomes (Bennett el al., 1992b). A defined function for hnRNP proteins has yet to be established, however there is increasing evidence that hnRNP proteins A1, C1/C2 and I/PTB contribute to efficiency and/ or regulation of splicing. The hnRNP A1 protein contains RRMs and a glycine rich domain (Query et al., 1989). The A1 protein has been observed to 21 preferentially bind the pyrimidine rich tract at the 3’ end of introns, (Swanson and Dreyfuss, 1988) which is suggested to aid in discriminating among pro-mRNAs and signal these differences to other components of the splicing complex (Lamrn et al., 1993). The A1 protein can also reanneal complementary RNA strands and this activity may alter the interaction of snRNAs with the pre-mRNA (Mayrand and Pederson, 1990). In vitro competition between A1 and SF2/ASF or 8035 has been shown to regulate choice of 5’ splice site of pre-mRNAs containing alternative 5’ splice sites (Mayeda et al., 1992). The hnRNP C1 and C2 proteins contain a single RRM domain (Swanson et al., 1987) and bind to poly(U) and to the U rich polypyrimidine region around the 3’ splice site of pre-mRNA and in downstream regions important for 3’ end cleavage and polyadenylation (Wilusz et al., 1990). A specific monoclonal that recognizes the C proteins (mAb 4F4) has been used to implicate C proteins in the splicing process. For example, addition of mAb 4F4 to an in vitro splicing reaction inhibits 5’ splice site cleavage but does not block spliceosome formation (Choi et al., 1986). The hnRNP I, also named PTB, protein contains RRM domains and can be specifically UV crosslinked to the polypyrimidine tract at the 3’ end of introns (Garcia et al., 1989; Gil et al., 1991; Patton et al., 1991). Unlike other hnRNP proteins which concentrate in the nucleoplasm, hnRNP/PTB also localizes in the perinuclear space (Ghetti et al., 1992). Antibodies directed against recombinant hnRNP I/PTB c0precipitate the spliceosome (Patton et al., 1991) and hnRNP I/PTB has been detected in purified spliceosomes (Bennett et al., 1992b; Garcia et al., 1989) implicating this protein to the splicing process. Immunodepletion and 22 complementation studies have shown that hnRNP I/PTB cannot restore splicing activity on its own but also requires a 100 kDa protein which has been shown to c0precipitate with hnRNP I/PTB along with a 33 kDa protein (Patton et al., 1991). HnRNP I/ PTB has been suggested to modulate alternative 3’ splice site selection although its exact role in splicing remains to be established (Mullen et al., 1991). SAPs A set of proteins termed SAPs (spliceosome associated proteins) associated with purified spliceosmes, have not been previously described as snRNP proteins or non- snRNP splicing factors (Bennett et al., 1992b). The SAPs have been shown to temporally bind to pre-mRNA during spliceosome assembly (Bennett et al., 1992b). A component of U5 snRNP, IBP (intron binding protein), binds to the 3’ end of introns and reacts with anti-Sm antibodies (Gerke and Steitz, 1986; Tazi et al., 1986). Immunodepletion of an 88 kDa protein (a putative SAP) from nuclear extracts arrested spliceosome assembly after formation of "A complex" and splicing activity could be restored by addition of affinity purified 88 kDa protein (Ast et al., 1991). The functional significance of most snRNP and non-snRNP associated proteins in pre-mRNA splicing is not known. Possible roles suggested include splice site selection, positioning of RNA and/or other protein components, stability, and participation in cleavage and/or ligation. Table 2 23 flammalian splicing factors A comprehensive list of mammalian non-snRNP splicing proteins, splicing factor and SAPs are shown. each factor and numbers of RRM (RNA recognition) Included is the size of and SR (Arginine-serine) motifs found in each factor. This table has been adapted from Table I in Lamm and Lamond, 1993. Protein Size (kDa) RRM SR UZAF55 53 3 + U2AF35 34 o + SF2/ASF 28 1 + SC-35 26 1 + SF3 ? not identified SF4A ? not identified SF4B ? not identified SF1* ? not identified SF2* 50 not identified SF3* ? not identified SF4* 110 not identified hnRNP A1 34 2 - hnRNP C1/C2 33/35 1 - hnRNP l/PTB 60 4 - IBP 100 not identified 88-kDa 88 not identified SAPS 155, 145, 130,115, not identified 114, 102, 92, 90, 88, 82, 72, 68, 62. 61, 60, 57, 55, 49, 42, 33 24 CONCLUSION: Nuclear galectins and pro-mRNA splicing: is there a connection? As previously stated, the major impetus for the research described in this thesis derived from the fact that galectin-3 appeared to be a component of nuclear RNP complexes. While nuclear RNPs are involved in various nuclear events relating to RNA biogenesis, their role in splicing has been unequivocally proved. The fact that mono- and disaccharides specific for galectin-3 perturbed in vitro pro-mRNA splicing intensified the need to prove a role for galectin-3 in splicing using the commonly accepted criteria of depletion and reconstitution as the standard for identification of splicing components. This thesis describes such experiments and defines galectin-3 as a new splicing factor. 25 BIBLIOGRAPHY Albrandt, K., Orida, N. K., and Liu, F.-T. (1987) Proc. Natl. Acad. Sci. USA. 84, 6859-6863. Amrein, H., Gorman, M. and Nothiger, R. (1988) Cell 55, 1025-1035. Arumugham, R. G., Hsieh, T. C.-Y., Tanzer, M. L., and Laine, R. A. (1986) Biochim. Biophys. Acta 883, 112-126. Arwal, N., Wang, J. 1. and Voss, P. G. (1989) J. Biol. Chem. 264, 17236-17242. Ast. G., Goldblatt, D., Offen, D., Sperling, J. and Sperling, R. (1991) EMBO J. 10, 425-432. Bandziulis, R. J., Swanson, M. S. and Dreyfuss, G. (1989) Genes Dev. 3, 431-437. Barabino, M. L., Blencowe, B. J., Ryder, U., Sproat, B. S. and Lamond, A. I. (1990) Cell 63, 293-302. Barondes, S. H. (1984) Science 223, 1259-1264. Barondes, S. H., Castronovo, V., Cooper, D. N. W., Cummings, R. D., Drickamer, K., Feizi, T., Gitt, M. A., Hirabyashi, J., Hughes, C., Kasai, K-I., Leffler, H., Liu, F-T., Lotan, R., Mercurio, A. M., Monsigny, M., Pillai, S., Poirer, F., Raz, A., Rigby, P. W. J., Rini, J. M. and Wang, J. L. (1994) Cell 76, 597-598. Bennett, M., Pinol-Roma, S., Staknis, D., Dreyfuss, G. and Reed, R. (1992a) Mol. Cell Biol 12, 3165-3175. Bennett, M., Michaud, 8., Kingston, J. and Reed, R. (1992b) Genes and Development 6, 1986-2000. Boggs, R. T., Gregor, P., Idriss, S., Belote, J. M. and Mckeown, M. (1987) Cell 50, 739-747. Bourgeois, C. A., Seve, A. P., Monsigny, M. and Hubert, J. (1987) Exp. Cell Res. 172, 365-376. Bouzon, M., Dussert, C., Lissitsky, J.-C., and Martin, P. M. (1990) Exp. Cell Res. 190, 47-56. Buvoli, M., Cobianchi, F. and Riva, S. (1992) Nuc. Acids Res. 20, 5017-5025. Carmo-Fonseca, M., Tollervey, D., Pepperkok, R., Barabino, S. M., Merdes, A., 26 Brunner, C., Zamore, P. D., Green, M. R., Hurt, E. and Lamond, A. I. (1991) EMBO J. 10, 195-206. Cerra, R. F., Gitt, M. A. and Barondes, S. H. (1985) J. Biol. Chem. 260, 10474-10477. Cherayil, B. J., Weiner, S. J. and Pillai, S. (1989) J. Exp. Med. 170, 1959-1972. Cherayil, B. J ., Chaitovitz, S., Wong, C. and Pillai, S. (1990) Proc. Natl. Acad. Sci. USA 87, 7324-7328. Choi, Y. D., Grabowski, P. J., Sharp P. A. and Dreyfuss, G. (1986) Science 231, 1534- 1539. Chou, T. B., Zachar, Z. and Bingham, P. M. (1987) EMBO J. 6, 4095-4104. Cooper, D. N. W. and Barondes, S. H. (1990) J. Cell Biol. 110, 1681-1691. Cooper, D. N. W., Massa, S. M., and Barondes, S. H. (1991) J. Cell Biol 115, 1437- 1448. Cowles, E. A., Agrwal, N., Anderson, R. L., and Wang, J. L. (1990) J. Biol. Chem. 265, 17706-17712. Crittenden, s. 1., Roff, c. F., and Wang, J. L. (1984) Mol. Cell. Biol 4, 1252-1259. Davis, L. I. and Blobel, G. (1986) Cell 45, 699-709. Dean, J. W., Chandrasekaran, S., and Tanzer, M. L. (1990)]. Biol. Chem 265, 12553- 12562. ' Drickamer, K. (1988) J. Biol. Chem. 263, 9557-9560. Eperon, I. C. and Krainer, A. R. (1993) In Hames, B. D. and Higgins, S. J. (eds), RNA Processing: A practical Approach. Facy, P., Seve, A. P., Hubert, M., Monsigny, M. and Hubert, J. (1990) Exp. Cell Res. 190, 151-160. Finlay, D. R., Newmeyer, D. D., Price, T. M. and Forbes, D. J. (1987) J. Cell Biol. 104, 189-200. Frigeri, L. G. and Liu, F.-T. (1992) J. Immuol. 148, 861-867. Fu, X.-D. and Maniatis, T. (1990) Nature 343, 437-440. 27 Fu, X.-D. and Maniatis, T. (1992) Science 256, 535-538. Fu, X.-D. and Maniatis, T. (1992) Proc. Natl. Acad. Sci. USA 89, 1725-1729. Fu, X.-D., Mayeda, A., Maniatis, T. and Krainer, A. R. (1992) Proc. Natl Acad. Sci. USA 89, 11224-11228. Fujiwara, S., Shinkai, H., Deutzmann, R., Pulsson, M., and Timpl, R. (1988) Biochem. J. 252, 453-461. Garcia, B. M., Jamison, S. F. and Sharp, P. A. (1989) Genes Dev. 3, 1874-1886. Ge, H. and Manley, J. L. (1990) Cell 62, 25-34. Ge, H., Zuo, P. and Manley, J. L. (1991) Cell 66, 373-382. Gerke, V. and Steitz, J. A. (1986) Cell 47, 973-984. Ghetti, A., Pinol-Roma, 8., Michael, W. M., Morandi, C. and Dreyfuss, G. (1992) Nuc. Acids Res. 20, 3671-3678. Gil, A., Sharp, P. A., Jamison, S. F. and Garcia, M. (1991) Gens Dev. 5, 1224-1236. Green, M. R. Rev. Cell Biol. (1991) 7, 559-599. Gritzmacher, C. A., Robertson, M. W., and Liu, F.-T. (1988)]. Immunol. 141, 2801- 2806. Guthrie, c. (1991) Science 253, 157-163. Hamann, K. K, Cowles, E. A., Wang, J. L., and Anderson, R. L. (1991) Exp. Cell Res. 196, 82-91. Hanover, J. A., Cohen, C. K., Willingham, M. C. and Park, M. K. (1987) J. Biol. Chem. 262, 9887-9894. Harper, J. E. and Manley, J. L. (1992) Gene Expression 2, 19-29. Harrison, F. L. (1991) J. Cell Sci. 100, 9-14. Hart, G. W., Haltiwanger, R. S., Holt, G. D. and Kelly, W. G. (1989) Annu. Rev. Biochem. 58, 841-874. Ho, M. K. and Springer, T. A. (1982)]. Immunol. 128, 1221-1228. 28 Holt, G. D., Snow, C. M., Senior, A., Haltiwanger, R. S., Gerace, L. and Hart, G. W. (1987)]. Cell Biol. 104, 1157-1164. Hubert, J., Seve, A. P., Bouvier, D., Masson, C., Bouteille, M. and Monsigny, M. (1985) Biol. Cell 55, 15-20. Hubert, J., Seve, A. P., Facy, P. and Monsigny, M. (1989) Cell Difi‘erentiation and Development 27, 69-81. Huflejt, M. E., Turck, C. W., Lindstedt, R., Barondes, S. H. and Leffler, H. (1993) J. of Biol. Chem. 268, 26712-26718. Jackson, S. P. and Tjian, R. (1988) Cell 55, 125-133. Jia, S. and Wang, J. (1988) J. Biol. Chem. 263, 6009—6011. Kenan, D. J ., Query, C. C. and Keene, J. d. (1991) Trends Biochem. Sci. 16, 214-220. Kljajic, 2., Schroder, H. C., Rottmann, M., Cuperlovic, M., Movsesian, M., Uhlenbruck, G., Gasic, M., Zahn, R. K. and Muller, W. E. G. (1987) Eur. J. Biochem. 169, 97-104. Knibbs, R. N., Perini, F., and Goldstein, I. J. (1989) Biochemistry 28, 6379-6392. Konarska, M. M. and Sharp, P. A. (1987) Cell 49, 763-774. Krainer, A. R. and Maniatis, T. (1985) Cell 42, 725-736. Krainer, A R., Conway, G. C. and Kozak, D. (1990) Genes Dev. 4, 1158-1171. Krainer, A. R., Mayeda, A., Kozak, D. and Binns, G. (1991) Cell 66, 383-394. Kramer, A., Frick, M. and Keller, W. (1987)]. Biol. Chem. 262, 17630-17640. Kramer, A., and Utans, U. (1991) EMBO 10, 1503-1509. Kumar, A. and Wilson, 8. H. (1990) Biochemistry 29, 10717-10722. Kurl, R. N., Holmes, S. C., Verney, E. and Sidransky, H. (1988) J. Biol. Chem. 27, 8974-8980. Laing, J. G. and Wang, J. L. (1988) Biochemistry 27, 5329-5334. Laing, J., Robertson, M., Gritzmacher, C., Wang, J. and Lui, F.-T. (1989) J. Biol. Chem. 264, 1907-1910. 29 Lamm, G. M. and Lamond, A. I. (1993) Biochirnica et Biophysica Acta 1173, 247-265. Lamond, A. I. (1993) BioEssays 15, 595-601. Leffler, H. and Barondes, S. H. (1986) J. Biol. Chem. 261, 10119-10126. Leffler, H., Masiarz, F. R., and Barondes, S. H. (1989) Biochemistry 28, 9222-9229. Lerner, M. R., and Steitz, J. A. (1979) Proc. Natl Acad. Sci USA. 76, 5495-5499. Lichtsteiner, S. and Schibler, U. (1989) Cell 57, 1179-1187. Liener, I. E., Sharon, N. and Goldstein, I. J ., eds (1986) Ann. Rev. Biochem. 55, 35-67. Lindstedt, R., Apodaca, G., Barondes, S., Mostov, K., and Leffler, H. (1991) J. Cell Biol. 115, 399a (Abstr. 2318). Liu, F.-T., Albrandt, K., Mendel, E., Kulczycki, A. and Orida, N. K. (1985) Proc. Natl. Acad. Sci. USA 82, 4100—4104. Luhrmann, R., Kastner, B. and Bach, M. (1990) Biochim. Biophys. Acta 1087, 265-292. Mancebo, R., Lo, P. C. and Mount, S. M. (1990) Mol Cell Biol. 10, 2492-2502. Maniatis, T. and Reed, R. (1987) Nature 325, 673-678. Massa, S. M., Copper, D. N. W., Leffler, H., and Barondes, S. H. (1992) J. Cell Biochem. Suppl 16D, 174 (Abstr. P420). Mattaj, I. W. (1988) In Structure and Function of Major and Minor Small Nuclear RNAs. M. Birnstiel, editor. Springer-Verlag. New York. Mayeda, A. and Krainer, A. R. (1992) Cell 68, 365-375. Mayeda, A., Zahler, A. M., Krainer, A. R. and Roth, M. B. (1992) Proc. Natl Acad. Sci. USA 89, 1301-1304. Mayrand, S. H. and Pederson, T. (1990) Nucleic Acids Res. 18, 3307-3318. Mercurio, A. M. and Shaw, L. M. (1991) Bioassays 13, 469-473. Meromsky, L., Lotan, R. and Raz, A. (1986) Cancer Res. 46, 5270-5275. Minoo, P., Sullivan, W., Solomon, L. R., Martin, T. E., Toft, D. 0., and Scott, R. E. (1989) J. Cell Biol 109, 1937-1946. 30 Monsigny, M., Kieda, C., and Roche, A. C. (1983) Biol Cell 47,95-110. Moutsatsos, I. K., Davis J. M., and Wang, J. L. (1986) J. Cell Biol 102, 477-483. Moutsatsos, I. K., Wade, M., Schindler, M. and Wang, J. (1987) Proc. Natl Acad. Sci. USA 84, 6452-6456. Mullen, M. P., Smith, c. W., Patton, J. c. and Nadal, G. B. (1991) Genes Dev. 5, 642- 655. Munroe, S. H., and Dong, X.-F. (1992) Proc. Natl Acad. Sci. USA 89, 895-899. Oda, Y., Leffler, H., Sakakura, Y., Kasai, K., and Barondes, S. H. (1991) Gene 99, 279-283. Olins, D. A., Olins, A. L., Seve, A. P., Bourgeois, C. A., Hubert, J. and Monsigny, M. (1988) Biol Cell 62, 95-98. Patton, J. G., Mayer, S. A., Tempst, P. and Nadal-Ginard, B. (1991) Genes Dev. 5, 1237-1251. Pontius, B. W. and Berg, P. (1990) Proc. Natl Acad. Sci USA 87, 8403-8407. Query, C. C., Bentley, R. C. and Keene, J. D. (1989) Cell 57, 89-101. Raz, A., Meromsky, L., Carmi, P., Karkash, R., Lotan, D., and Lotan, R. (1984) EMBO J. 3, 2979-2983 Raz, A., Meromsky, L. and Lotan, R. (1986) Cancer Res. 46, 3667-3672. Raz, A., Pazerini, G. and Carmi, P. (1989) Cancer Res. 49, 3489-3493. Raz, A., Carmi, P., Raz, T., Hogan, V., Mohammed, A., and Wolman, S. R. (1991a) Cancer Res. 51, 2173-2178. Raz, A., Zhu, D., Hogan, V., Shah, N., Raz, T., Karkash, R., Pazerini, G. and Carmi, P. (1991b) Int. J. Cancer 46, 871- Reddy, R. (1986) Nucl Acids Res. 14, r61-r72. Reeves, R. and Chang, C. (1983) J. Biol Chem. 258, 679-687. Rio, D. C. (1992) Curr. Opin. Cell Biol 4, 444-452. Robertson, M. W., Albrandt, K., Keller, D., and Liu, F. T. (1990) Biochemistry 29, 31 8093-8100. Roff, C. F. and Wang, J. L. (1983) J. Biol Chem. 258, 10657-10663. Ruskin, B., Zamore, P. D. and Green, M. R. (1988) Cell 52, 207-219. Sato, S., and Hughes, R. C. (1992) J. Biol Chem. 267, 6983-6990. Schroder, H. C., Becker, R., Bachmann, M., Gramzow, M., Seve, A.-P., Monsigny, M. and Muller, W. E. G. (1986) Biochim. Biophys. Acta 868 108-118. Schroder, H. C., Facy, P., Monsigny, M., Pfeifer, K., Bek, A., and Muller, W. E. G. (1992) Eur. J. Biochem. 205, 1017-1025. Seve, A. P., Hubert, J ., Bouvier, D., Bouteille, M. and Monsigny, M. (1985) Exp. Cell Res. 157, 533-538. Seve, A. P., Hubert, J., Bouvier, D., Bourgeois, C., Midoux, P., Roche, A. C. and Monsigny, M. (1986) Proc. Natl Acad. sci. USA 83, 5997-6001. Seve, A. P., Felin, M., Moyne, M. A. D., Sahraoui, T., Aubry, M. and Hubert, J. (1993) Glycobiology 3, 23-30. Sharon, N. and Lis, H. eds (1989) Lectins (Chapman and Hall, London). Sharp, P. A. (1987) Science 235, 766-771. Soulard, M., Barque, J.-P., Valle, V. D., Hernandez-Verdun, H., Masson, C., Danon, F., and Larsen, C.-J. (1991). Exp. Cell Res. 193, 59-71. Sparrow, C. P., Leffler, H. and Barondes, S. H. (1987) J. Biol Chem. 262, 7383-7390. Spector, D. L., Fu, X. D. and Maniatis, T. (1991) EMBO J. 10, 3467-3481. Sparrow, C. P., Leffler, H., and Barondes, S. H. (1987) J. Biol Chem. 262, 7383-7390. Swanson, M. S., Nakagawa, T. Y., LeVan, K. and Dreyfuss, G. (1987) Mol Cell Biol 7, 1731-1739. Swanson, M. S. and Dreyfuss, G. (1988) EMBO J. 11, 3519-3529. Tazi, J., Alibert, C., Temsarnini, J., Reveillaud, I., Cathala, G., Brunei, C. and Jeanteur, P. (1986) Cell 47, 755-766. Tracy, B. M., Feizi, T., Abbott, W. M., Carruthers, R. A., Green, B. N., and Lawson, 32 A. M. (1992) J. Biol Chem. 267, 10342-10347. Utans, U. and Kramer, A. (1990) Methods Enzymol 181, 3-19. Vellard, M., Sureau, A., soret, J., Martinerie, C. and Perbal, B. (1992) Proc. Natl Acad. Sci. USA 89, 2511-2515. Voelker, R. A., Gibson, W., Graves, J. P., Sterling, J. F. and Eisenberg, M. T. (1991) Mol Cell Biol 11, 894-905. Wang, J. I..., Laing, J. G., and Anderson, R. L. (1991) Glycobiology 1, 243-252. Wang, J. L., Werner, E. A., Laing, J. G. and Patterson, R. J. (1992) Trans. Biochem. Soc. 20, 269-274. Wasano, K, Hirakawa, Y., and Yamamoto, T. (1990) Cell Tissue Res. 259, 43-49. Weis, W. I., Kahn, R., Fourrne, R., Drickamer, K., and Hendrickson, W. A. (1991) Science 254, 1608-1615. Wilusz, J. and Shenk, T. (1990) Mol Cell Biol 10, 6397-6407. Woo, H. J., Shaw, L. M., Messier, J. M. and Mercurio, A. M. (1990) J. Biol Chem. 265, 7097-7099. Woo, H. J., Lotz, M. M., Jung, J. U., and Mercurio, A. M. (1991)]. Biol Chem. 266, 18419-18422. Yoneda, Y., Imamoto-Sonobe, N., Yamaizumi, M. and Uchida, T. (1987) Exp. Cell Res. 173, 173, 586-595. Zamore, P. D. and Green, M. R. (1989) Proc. Natl Acad. Sci. U.S.A. 86, 9243-9247. Zamore, P. D. and Green, M. R. (1991) EMBO 10, 207-214. Zamore, P. D., Patton, J. G. and Green, M. R. (1992) Nature 355, 609-614. Zhang, M., Zamore, P. D., Carmo, F. M., Lamond, A. I. and Green, M. R. (1992) Proc. Natl Acad. Sci USA 89, 8769-8773. Zhou, Q., and Cummings, R. D. (1990) Arch. Biochem. Biophys. 281, 27-35. CHAPTER II IDENTIFICATION OF GALECFIN-3 AS A REQUIRED FACTOR IN pro-mRNA SPLICING (CBP35/lectin) Sue F. Dagher', John L. Wang’ and Ronald J. Patterson§ The Departments of Microbiology’ and Biochemistry“ and the Genetics Program', Michigan State University, East Lansing, MI 48824 33 34 FOOTNOTES Acknowledgement We thank Richard Schwartz and Betty Werner in the Department of Microbiology for helpful discussions and members of the Wang laboratory for their invaluable assistance. This work was supported by grants MCB 91-22363 from the National Science Foundation (R.J.P.) and GM-38740 from the National Institutes of Health (J.LW.). 35 ABSTRACT Galectin-3 (Mr ~35,000) is a galactose/lactose specific lectin found in association with ribonucleoprotein complexes in many animal cells. Cell-free splicing assays have been carried out to study the requirement of galectin-3 in RNA processing by HeLa cell nuclear extracts using 32P-labeled MINX as pro-mRNA substrate. Addition of saccharides that bind galectin-3 with high affinity inhibited product formation in the splicing assay; parallel addition of carbohydrates that do not bind to the lectin failed to yield the same result. Nuclear extracts depleted of galectin-3 by affinity adsorption on a lactose-agarose column were deficient in splicing. Extracts subjected to parallel adsorption on control cellobiose-agarose remained splicing competent. The activity of the galectin-3-depleted extract could be reconstituted by the addition of purified recombinant galectin-3 whereas the addition of other lectins, either with a similar saccharide binding specificity (soybean agglutinin) or with a different specificity (wheat germ agglutinin), did not restore splicing activity. The formation of splicing complexes was also sensitive to galectin-3 depletion and reconstitution. Together, these results define a requirement for galectin-3 in pre-mRNA splicing and identify it as a new splicing factor. 36 INTRODUCTION Galectin-3 (Barondes et al., 1994) is the new name for the galactose(Gal)/lactose(Lac) specific lectin previously known under a number of different designations, including Carbohydrate Binding Protein 35 (CBP35) (J ia and Wang, 1988), Mac-2 (Cherayil et al., 1989), IgE-binding protein (Albrandt et al., 1987), CBP30 (Sato et al., 1993), L-29 (Leffler et al., 1989), and L-34 (Raz et al., 1989). In this communication, we will use the designation galectin-3 when we refer to the gene / protein in general, assuming that studies carried out on the gene/protein under any one of the above names is applicable to all of them. There are instances, however, in which the specific molecule used by one laboratory is slightly (but of significance) different then the corresponding molecule of another laboratory (e.g., the cDNAs reported for murine CBP35, Mac-2, and L-34 are of different lengths). In those cases, we will use the old designation to highlight the specific source of the molecule. The galectin family of animal lectins is distinguished by the Gal/Lac specificity of its carbohydrate recognition domain (CRD), with highly conserved residues between members of the family (galectin-1, -2, -3, and -4) and between the homologues found in various species for any given single member (for reviews, see Anderson and Wang, 1992 and Hirabayashi and Kasai, 1993). The polypeptide of galectin-3 is delineated into two domains (Anderson and Wang, 1992; J ia and Wang, 1988): an amino-terminal half that is Pro and Gly rich, with limited homology to proteins of the heterogeneous nuclear ribonucleoprotein complexes and a carboxyl- terminal half homologous to the CRD of other members of the galectin family. 37 Another distinguishing feature of the galectin family is that all members have been found both inside cells as well as at the cell surface. For galectin-3, the majority of the protein is found in the cytoplasm and nucleus of mouse 3T3 fibroblasts, in the form of ribonucleoprotein (RNP) complexes (Laing and Wang, 1988; Wang et al., 1992). For example, treatment of permeabilized cells with ribonuclease A released the lectin from the nucleus, with loss of immunofluorescent staining. Two isoelectric species of galectin-3 have been identified: a phosphorylated (pI 8.2) and a non- phosphorylated (pI 8.7) form (Cowles et al., 1990). The phosphorylated species is found in both the nucleus and cytoplasm whereas the nonphosphorylated form localizes exclusively in the nucleus. Nuclear extracts (NE), prepared from HeLa cells and capable of carrying out pro-mRNA splicing, contain galectin-3. On the basis of preliminary experiments that showed saccharide ligands with high affinity for galectin-3 can perturb the splicing reaction, we sought conditions to deplete galectin-3 from nuclear extracts. We now report that extracts depleted of galectin-3 do not support spliceosome formation or pre-mRNA splicing. The galectin-3 depleted extracts, however, regain splicing activity upon reconstitution with the recombinant lectin purified from an E. coli expression system. MATERIALS AND METHODS Preparation of Nuclear Extracts and Their Depletion and Reconstitution. NE were prepared from HeLa S3 suspension cultures in buffer D (20 mM Hepes (pH 7.9), 20% (v/v) glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM phenyl methylsulfonyl 38 fluoride (PMSF), and 0.5 mM (DTT)) as described (Dignam et al., 1983). Typically, NE at protein concentrations of approximately 18-22 mg/ ml were prepared and stored as frozen aliquots at -80°C. Protein concentrations were determined by Bradford assay (Bradford, 1976, from BioRad). NE were depleted of carbohydrate binding proteins on the basis of their binding to a-Lac insolubilized on 6% beaded agarose (LAC-A), purchased from Sigma. Agarose derivatized with cellobiose (CELLO-A) was used as a control for depletion. 200-250 [11 of packed agarose were washed with 20 volumes of wash buffer (20 mM Hepes (pH 7.9), 0.5 M NaCl) in disposable columns (Pierce). NE were preincubated in buffer 1 (the composition of buffer 1 corresponds to 60% buffer D) containing, in addition, 2.5 mM MgC12, 1 mM ATP, 5 mM creatine phosphate in a vol of 50 11.1 for 30 min at 30°C. NaCl was then added to a final concentration of 0.5 M. Preincubated NE were then added to the washed saccharide-agarose and incubated for 30 min at 4°C with rotation. The unbound (UB) fraction was removed from the column. The matrices were next washed with 50 pl of buffer 1 containing 0.5 M NaCl and this wash was added to the UB fraction. The matrices were washed with 10 volumes of wash buffer and the bound (B) material eluted by boiling in 200 pl of Laemrnli sample buffer (Laemmli, 1970). Aliquots (20 ul) of nondepleted NE and the UB fractions of the saccharide-adsorption procedure were dialyzed in a microdialyzer (Pierce) for 40 min against buffer 1. In reconstitution experiments, recombinant CBP35 (rCBP35) (Agrwal et al., 1993) or other lectins (EY Laboratories) were added to the NE or UB fractions prior to dialysis. The dialyzed fractions were then assayed for splicing activity or splicing 39 complex formation. Splicing Assay. The plasmid used to transcribe the MINX pre-mRNA substrate (Zillmann et al., 1988) was a kind gift of Dr. Susan Berget (Baylor College of Medicine). The MINX pre-mRNA was labeled with [32P]GTP during Sp6 polymerase (Gibco) transcription and the monomethyl cap was added during transcription (Zillmann et al., 1988). Splicing reactions (10 ul) contained NE (3 1:1) or dialyzed UB or dialyzed non- depleted NE (8 111) and 2.5 mM MgClz, 1.5 mM ATP, 20 mM creatine phosphate, 0.5 mM DTT and 20 units RNasin. The final protein concentration of the dialyzed extracts was 7-12 mg/ml. For reactions lacking ATP, both ATP and creatine phosphate were omitted. In experiments testing the effect of exogenously added carbohydrates, the NE were preincubated with saccharides at 30°C for 30 min prior to the addition of 32P-labeled pre-mRNA. Splicing reactions were carried out at 30°C for 45 min. Each splicing sample was diluted to 100 ill with 125 mM Tris (pH 6.8), 1 mM EDTA, 0.3 M sodium acetate. Proteinase K was added to a final concentration of 2 mg/ml and the sample digested for 1 hr at 37°C. RNA was extracted with 200 pl phenol-chloroform-isoamyl alcohol (50:50:1 (v/v)) followed by 200 pl chloroform. RNAs were precipitated with 300 111 ethanol in a dry ice ethanol bath. The extracted RNAs were subjected to electrophoresis in 13% polyacrylamide (bisacrylamide:acrylamide, 0.8:50 (w/w)) - 8.3 M urea gels, followed by autoradiography. The intensities of the bands on gels were quantitated by direct B-counting using an AMBIS system. The percent product formation (ligated exon 1-ex0n 2) was 40 calculated by dividing the radioactivity in the product by the total radioactivity in the pre-mRNA substrate, the splicing intermediates (exon 2-lariat, excised lariat, exon 1) and the product. Gel Mobility Shin Assay for Splicing Complex Formation. The formation of splicing complexes was monitored by native gel electrophoresis (Konarska and Sharp, 1986). Splicing reactions (10 pl) were incubated at 30°C for 15-20 min. Heparin was added (final concentration 0.6 mg/ml) and the mixtures were incubated at 30°C for 15 min. Before electrophoresis, 1 pl of glycerol (containing 0.2% each of bromphenol blue, xylene cyanole and phenol red) was added to each sample. The samples were loaded onto a pro-run polyacrylamide gel (bisacrylamidezacrylamide, 1:80 (w/w)) and electrophoresis was carried out in 0.5 M Tris base - 0.5 M glycine (pH 8.8) at 4°C, 25 V/cm for 105 min. The migration of splicing complexes was determined by autoradiography. Western Blot Analysis. Polyacrylamide gel electrophoresis in SDS was carried out in 12% acrylamide gels (Laemmli, 1970). The separated components were transferred to PVDF membrane (BioRad) in 25 mM Tris, 193 mM glycine and 20% methanol. Following transfer, the membranes were incubated with 5% nonfat dry milk dissolved in phosphate buffered saline containing 0.05% Tween 20 (PBS-T). Galectin-3 was detected using the rat monoclonal antibody, anti-Mac 2 (Ho and Springer, 1982). Incubation with the primary antibody (freshly diluted 1:5,000 in 5% nonfat dry milk - PBS-T) was carried out at room temperature for 2 hr, followed by five washes in PBS-T, 15 min each. The membranes were incubated with horseradish peroxidase conjugated goat anti-rat antibodies (Pierce) (diluted 1:3000 in PBS—T) for 41 30 min at room temperature followed by five 15 min washes in PBS-T. The products of the horseradish peroxidase reaction were revealed with the ECL reagents from Amersham, following the manufacturer’s protocol. RESULTS Effect of Saccharides on pro-mRNA Splicing. The cell-free assay for the splicing of pro-mRNA by NE of HeLa cells was optimized for the MINX substrate. In a typical assay (final protein concentration of reaction ~10 mg/ ml), approximately 20- 30% product formation was observed (Fig. 1, lane 1). This conversion showed a stringent requirement for ATP (Fig. 1, lane 14). When the NE was preincubated with saccharides (75 mM) prior to the addition of the pre-mRNA substrate, inhibition of product formation was observed for Lac, thiodigalactoside (TDG), and melibiose (Fig. 1, lanes 2-4, respectively). Other mono- and disaccharides tested, as well as the non-carbohydrate inositol, failed to show inhibition of product formation (Fig. 1, lanes 5-13). The effect of the inhibitory saccharides was concentration dependent; half- maximal inhibition were observed at <50 mM for TDG and Lac (Fig. 2, lanes 3-6 and lanes 7-10, respectively). Both of these saccharides bind to galectin-3 (Leffler and Barondes, 1986) with approximately the same hierarchy of affinities as those seen in the inhibition of splicing. The disaccharide cellobiose (Fig 2, lanes 15-18) and the monosaccharide Gal (Fig. 2, lanes 11-14) did not show an effect on the cell free splicing assay. Although Gal is a saccharide ligand for the galectin family of lectins, its binding affinity for galectin-3 is about two orders of magnitude lower than the 42 Figure l The effect of saccharides on pre-mRNA splicing. Splicing reactions were incubated in the absence of saccharide (lane 1) or in the presence of 75 mM various compounds (lanes 2-13) or in the absence of ATP (lane 14). The positions of migration of pre-mRNA, splicing intermediates and product are indicated on the right. 43 B CD 3 e 8mm OJ E a) 0- — <0 — to (0.13") >‘ 0- Em_go.98$g86£o8+— +8119OQGoC’t‘mmgfiE moEmmamfigotogw 1 (A. 12 3 45 67‘8 91011121314 Figure 1 44 affinity of Lac (Leffler and Barondes, 1986). Thus, the inhibitory versus noninhibitory saccharides observed in the splicing assay correlate with the relative order of their binding affinities for galectin-3. The inhibitory effects of Lac and TDG were neither extract nor pre-mRNA substrate restricted. NE prepared from HeLa cells over the course of two years were sensitive to inhibition by these saccharides. In addition, similar splicing inhibition was observed using human B-globin pre-mRNA (Krainer et al., 1984) and another recombinant adenovirus transcript pRSP—l (Konarska et al., 1984). For example, a human serum albumin conjugate containing 14 covalently linked blood group A- tetrasaccharide moieties completely inhibited the splicing of the B-globin substrate at a concentration as low as 170 pM while a parallel serum albumin control yielded no effect. Blood group A-tetrasaccharide has been shown to exhibit significantly greater affinity for galectin-3 than simple mono- and disaccharides ligands (Leffler and Barondes, 1986; Sparrow et al., 1987). All of these results suggest that certain saccharides can perturb cell-free splicing, possibly by binding to galectin-3 in the NE. Splicing Activity of Galectin-3-Depleted Extracts. To test for a role of nuclear galectin-3 in pre-mRNA splicing, we used saccharide affinity adsorption to deplete the protein from NE. NE were pretreated with 0.5 M NaCl to dissociate splicing and/ or RNP complexes and then incubated with LAC-A. Parallel experiments were carried out on CELLO-A as a control matrix. To monitor the extent of depletion, the levels of galectin-3 in the original NE and in the UB and B fractions of the saccharide adsorptions were quantitated using the galectin-3 specific monoclonal antibody, anti-Mac-2 (Ho and Springer, 1982; Cherayil et al., 1989). A comparison 45 Figure 2 Concentration dependence of the effect of TDG and lac on pre-mRNA splicing. Splicing reactions were incubated in the presence 50, 75, 125 and 150 mM Lac (lanes 3-6), TDG (lanes 7-10), Gal (lanes 11-14) and Cello (lanes 15-18) as described in Materials and Methods. Control splicing reactions were incubated in the absence (lane 1) or presence (lane 2) of ATP and creatine phosphate. The values shown at the bottom indicate percent product formation. TDG Lw %I mm Q "‘- .. .... ‘1’21 12 3456 78910 1112131415161718 290 6310 4220 21262115 22222322 %product Figure 2 47 of the UB and B fractions showed that LAC-A removed >95% of galectin-3 (Fig. 3A, lanes 1 and 2). Control CELLO-A removed <5% of galectin-3 (Fig. 3A, lanes 3 and 4). Protein determinations revealed that the depletion procedure also decreased the total protein content of the UB fractions by about 25%, compared to the non-treated NE. Thus, when the saccharide affinity-depleted extracts were tested in splicing assays, the non-treated extracts were diluted appropriately for comparison. The UB fraction of the LAC-A adsorption, depleted of galectin-3, exhibited little or no splicing activity (Fig. 3B, lane 3), when compared to the corresponding fraction of the CELLO-A adsorption (Fig. 3B, lane 4) or to the original NE (Fig. 3B, lane 2). CELLO-A depleted extracts showed no change in splicing activity. Thus, the removal of galectin-3 from NE resulted in a complete loss of splicing activity. Reconstitution of Splicing in Galectin-3-Depleted Extracts. We had previously described the production of a recombinant galectin-3 using an E. coli expression system for the cloned cDNA for CBP35 (Agrwal et al., 1993). The availability of this recombinant protein, designated here as rCBP35, provided the unique opportunity to test whether splicing activity in the UB fraction of LAC-A adsorption can be reconstituted by addition of a single, purified protein. Indeed, the addition of rCBP35 restored splicing activity to the galectin-3 depleted extract (Fig. 4, lanes 1 and 2). In contrast, the plant lectin soybean agglutinin, with a saccharide binding specificity (Sharon and Lis, 1989) similar to that of galectin-3, was unable to reconstitute splicing activity (Fig 4, lane 3). Similarly, wheat germ agglutinin, which binds to glycoconjugates containing sialic acids and/or N-acetyl-D-glucosamine (Sharon and Lis, 1989) also failed to restore splicing activity to the galectin-3 48 Figure 3A Comparison of the levels of galectin-3 in NE and in the U8 and B fractions when NE were subjected to adsorption on LAC-A and CELLO-A. NE were prepared for saccharide affinity chromatography as described in Materials and Methods. B and U3 proteins were separated by SDS-PAGE then analyzed by Western blotting with anti-Mac—Z monoclonal antibody. Lanes 1 and 2, UB and B fractions, respectively, from LAC-A; lanes 3 and 4, UB and B fractions, respectively, from CELLO-A and; lane 5, non-depleted NE. 49 Figure 3A 50 Figure 38 Comparison of splicing activity of NE and the UB fractions of LAC- and CELLO- affinity adsorptions. Lanes 1 and 2, nondepleted control splicing reactions in the absence and presence of ATP, respectively; lane 3, LAC-A UB fraction and lane 4, CELLO-A UB fraction. 1 . 5 <-O._._m0 m: «.05 m3 mh<+.mz n_._.<- m_Z H Figure 3B 52 depleted extract (data not shown). Finally, neither rCBP35 (Fig. 4, lane 4) nor soybean agglutinin (data not shown) had an effect on the splicing assay when added to a non-depleted NE. The restoration of splicing activity by rCBP35 to the UB fraction of the LAC-A adsorption was dependent on the amount of recombinant protein added. Addition of 0.5 pg rCBP35 did not restore activity (Fig 5A, lane 2) whereas 1 to 4 pg rCBP35 reconstituted activity (Fig 5A, lanes 3-5). Surprisingly, addition of 8 pg of rCBP35 resulted in no splicing activity (Fig. 5A, lane 6). The amount of rCBP35 added in the reconstitution experiments should be put in context of the total protein content in a reconstituted splicing reaction (~60 pg) and of galectin-3 (0.5-1 ng) in an equivalent splicing reaction containing non-depleted NE. The maximal extent of reconstituted splicing activity represented >70% of the activity of non-depleted NE. More importantly, galectin-3 depleted extracts reconstituted with rCBP35 showed inhibition of splicing upon addition of TDG, as was observed with the original NE (Fig 5A, lane 7). Analysis of Spliceosome Complex Formation in Original, Depleted, and Reconstituted Extracts. The MINX pro-mRNA forms several complexes during the course of the splicing reaction, as revealed by gel mobility shift assays of the 32P- labeled substrate in non-denaturing gels (Konarska and Sharp, 1986). The first complex formed is an ATP-independent complex designated as the H complex. The addition of ATP results in the association of U1 and U2 snRNPs, forming the A and A’ complexes, respectively. This, in turn, is followed by the association of U4, U5 and U6 snRNPs to yield the B complex (Zillmann et al., 1988). 53 Figure 4 The effect of rCBP35 and soybean agglutinin on the splicing activity of the UB fraction of LAC-A. Lanes 1-3, splicing in LAC-A UB fraction and lane 4, splicing in non-depleted extracts. Lane 1, no addition; lanes 2 and 4,addition of rCBP35 (2.0 pg); lane 3 addition of soybean agglutinin (2.0 pg). 4 mmdmoimz. 1234 A