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 for galectin-3 in RNA processing by HeLa cell nuclear extracts by using 32P-labeled MINX as the pre-mRNA substrate. Addition of saccharides that bind galectin-3 with high affinity inhibited product formation in the splicing assay, while addition of carbohydrates that do not bind to the lectin did not inhibit product formation. Nuclear extracts depleted ofgalectin-3 by affinity adsorption on a lactoseagarose column were deficient in splicing activity. Extracts subjected to parallel adsorption on control celiobiose-agarose retained splicing activity. 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 splicing factor.Galectin-3 (1) is the name for the galactose(Gal)/lactose (Lac)-specific lectin previously known under a number of different designations, including carbohydrate binding protein 35 (CBP35) (2), Mac-2 (3), IgE-binding protein (4), CBP30 (5), L-29 (6), and L-34 (7). In this communication, we will use the designation galectin-3 when we refer to the gene or protein in general, assuming that studies carried out on the gene or 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 significantly) different from the corresponding molecule of another laboratory-e.g., the cDNAs reported for murine CBP35, Mac-2, and L-34 are of different lengths. In this case, 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 homologs found in various species for any given single member (for reviews see refs. 8 and 9). The polypeptide of galectin-3 is delineated into two domains (2,8): an N-terminal half that is proline and glycine rich, with limited similarity to proteins of the heterogeneous nuclear ribonucleoprotein (RNP) complexes, and a C-terminal half that is similar to the CRD of other members of the galectin family. The majority of galectin-3 is found in the cytoplasm and nucleus of mouse 3T3 fibroblasts in the form of RNP complexes (10, 11). For example, treatment of permeabilized cells with ribonuclease A released the lectin from the nucleus with concomitant los...

Galectins are a family of ␤-galactoside-binding proteins that contain characteristic amino acid sequences in the carbohydrate recognition domain (CRD) of the polypeptide. The polypeptide of galectin-1 contains a single domain, the CRD. The polypeptide of galectin-3 has two domains, a carboxyl-terminal CRD fused onto a proline-and glycine-rich amino-terminal domain. In previous studies, we showed that galectin-3 is a required factor in the splicing of nuclear pre-mRNA, assayed in a cell-free system. We now document that (i) nuclear extracts derived from HeLa cells contain both galectins-1 and -3; (ii) depletion of both galectins from the nuclear extract either by lactose affinity adsorption or by double-antibody adsorption results in a concomitant loss of splicing activity; (iii) depletion of either galectin-1 or galectin-3 by specific antibody adsorption fails to remove all of the splicing activity, and the residual splicing activity is still saccharide inhibitable; (iv) either galectin-1 or galectin-3 alone is sufficient to reconstitute, at least partially, the splicing activity of nuclear extracts depleted of both galectins; and (v) although the carbohydrate recognition domain of galectin-3 (or galectin-1) is sufficient to restore splicing activity to a galectin-depleted nuclear extract, the concentration required for reconstitution is greater than that of the full-length galectin-3 polypeptide. Consistent with these functional results, double-immunofluorescence analyses show that within the nucleus, galectin-3 colocalizes with the speckled structures observed with splicing factor SC35. Similar results are also obtained with galectin-1, although in this case, there are areas of galectin-1 devoid of SC35 and vice versa. Thus, nuclear galectins exhibit functional redundancy in their splicing activity and partition, at least partially, in the nucleoplasm with another known splicing factor.Galectins are a family of widely distributed proteins that (i) bind to ␤-galactosides and (ii) contain characteristic amino acid sequences in the carbohydrate recognition domain (CRD) of the polypeptide. At present, eight mammalian galectins have been reported and classified into three subgroups, according to the content and organization of the domains in their respective polypeptides (for reviews, see references 3 and 21). The prototype subgroup consists of polypeptides (ϳ14 kDa) with a single domain, the CRD. Galectins-1, -2, -5, and -7 are members of this subgroup. Another subgroup is the tandem repeat type, which has three members: galectins-4, -6, and -8. These galectins have two domains, each a CRD, connected by a linker region. Finally, the chimera subgroup is, at present, represented by a single member, galectin-3. Its polypeptide contains two domains, a CRD fused onto a Pro-, Gly-rich domain.In previous studies, we reported the localization of galectin-3 to the cell nucleus in the form of a ribonucleoprotein (RNP) complex (23, 29). We also identified it as one of the many proteins required for the splicing of pre-mRNA, ...

This review summarizes selected studies on galectin-3 (Gal3) as an example of the dynamic behavior of a carbohydrate-binding protein in the cytoplasm and nucleus of cells. Within the 15-member galectin family of proteins, Gal3 (Mr ~30,000) is the sole representative of the chimera subclass in which a proline- and glycine-rich NH2-terminal domain is fused onto a COOH-terminal carbohydrate recognition domain responsible for binding galactose-containing glycoconjugates. The protein shuttles between the cytoplasm and nucleus on the basis of targeting signals that are recognized by importin(s) for nuclear localization and exportin-1 (CRM1) for nuclear export. Depending on the cell type, specific experimental conditions in vitro, or tissue location, Gal3 has been reported to be exclusively cytoplasmic, predominantly nuclear, or distributed between the two compartments. The nuclear versus cytoplasmic distribution of the protein must reflect, then, some balance between nuclear import and export, as well as mechanisms of cytoplasmic anchorage or binding to a nuclear component. Indeed, a number of ligands have been reported for Gal3 in the cytoplasm and in the nucleus. Most of the ligands appear to bind Gal3, however, through protein-protein interactions rather than through protein-carbohydrate recognition. In the cytoplasm, for example, Gal3 interacts with the apoptosis repressor Bcl-2 and this interaction may be involved in Gal3’s anti-apoptotic activity. In the nucleus, Gal3 is a required pre-mRNA splicing factor; the protein is incorporated into spliceosomes via its association with the U1 small nuclear ribonucleoprotein (snRNP) complex. Although the majority of these interactions occur via the carbohydrate recognition domain of Gal3 and saccharide ligands such as lactose can perturb some of these interactions, the significance of the protein’s carbohydrate-binding activity, per se, remains a challenge for future investigations.

In previous studies we showed that galectin-1 and galectin-3 are factors required for the splicing of pre-mRNA, as assayed in a cell-free system. Using a yeast two-hybrid screen with galectin-1 as bait, Gemin4 was identified as a putative interacting protein. Gemin4 is one component of a macromolecular complex containing approximately 15 polypeptides, including SMN (survival of motor neuron) protein. Rabbit anti-galectin-1 co-immunoprecipitated from HeLa cell nuclear extracts, along with galectin-1, polypeptides identified to be in this complex: SMN, Gemin2 and the Sm polypeptides of snRNPs. Direct interaction between Gemin4 and galectin-1 was demonstrated in glutathione S-transferase (GST) pull-down assays. We also found that galectin-3 interacted with Gemin4 and that it constituted one component of the complex co-immunoprecipitated with galectin-1. Indeed, fragments of either Gemin4 or galectin-3 exhibited a dominant negative effect when added to a cell-free splicing assay. For example, a dose-dependent inhibition of splicing was observed in the presence of exogenously added N-terminal domain of galectin-3 polypeptide. In contrast, parallel addition of either the intact galectin-3 polypeptide or the C-terminal domain failed to yield the same effect. Using native gel electrophoresis to detect complexes formed by the splicing extract, we found that with addition of the N-terminal domain the predominant portion of the radiolabeled pre-mRNA was arrested at a position corresponding to the H-complex. Inasmuch as SMN-containing complexes have been implicated in the delivery of snRNPs to the H-complex, these results provide strong evidence that galectin-1 and galectin-3, by interacting with Gemin4, play a role in spliceosome assembly in vivo.

Although the Ul small nuclear ribonucleoprotein particle (snRNP) was the first mRNA-splicing cofactor to be identified, the manner in which it functions in splicing is not precisely understood. Among the information required to understand how Ul snRNP participates in splicing, it will be necessary to know its structure. Here we describe the in vitro reconstitution of a particle that possesses the properties of native Ul snRNP. 32P-labeled Ul RNA was transcribed from an SP6 promoter-human Ul gene clone and incubated in a HeLa S100 fraction. A Ul particle formed which displayed the same sedimentation coefficient (-1OS) and buoyant density (1.40 g/cm3) as native Ul snRNP. The latter value reflects the ability to withstand isopycnic banding in Cs2SO4 without prior fixation, a property shared by native Ul snRNP. The reconstituted Ul particle reacted with both the Sm and RNP monoclonal antibodies, showing that these two classes of snRNP proteins were present.Moreover, the reconstituted Ul snRNP particle was found to display the characteristic Mg2+ switch of nuclease sensitivity previously described for native Ul snRNP: an open, nuclease-sensitive conformation at a low Mg2+ concentration (3 mM) and a more compact, nuclease-resistant organization at a higher concentration (15 mM). The majority of the Ul RNA in the reconstituted particle did not contain hypermethylated caps, pseudouridine, or ribose 2-0-methylation, showing that these enigmatic posttranscriptional modifications are not essential for reconstitution of the Ul snRNP particle. The extreme 3' end (18 nucleotides) of Ul RNA was required for reconstitution, but loop II (nucleotides 64 to 77) was not. Interestingly, the 5' end (15 nucleotides) of Ul RNA that recognizes pre-mRNA 5' splice sites was not required for Ul snRNP reconstitution.Ul RNA is one of several small nuclear RNAs that participate in mRNA splicing (21). Ul RNA is associated with at least nine proteins in a complex known as the Ul small nuclear RNP particle (snRNP) (3, and references cited therein). Several of these proteins are common to snRNPs that contain U2, U5, or U4/U6 RNAs, but three are specific to Ul snRNP (2,3,10,11,30,36).The detailed structure of the Ul snRNP has not been determined. Nuclease digestion studies of human Ul, U2, U4, and U5 snRNPs have revealed a major protected region containing the sequence A(U),G, where n is 2 3, flanked by stem-loop structures (17). This is the binding site for snRNP proteins reactive with Sm antibodies and is therefore known as the Sm binding site or Sm domain (14,17,23). The structure of the Ul snRNP is Mg2> dependent: at high Mg2> concentrations (>7 mM), the particle undergoes a switch to a more compact conformation that renders additional regions of the Ul RNA nuclease resistant (18,32).In this paper we report that under appropriate conditions, it is possible to assemble a particle that possesses several properties of native Ul snRNP, including the characteristic Mg2+-dependent conformational switch. We also define some of the RNA sequences requi...