Association of galectin-1 and galectin-3 with Gemin4 in complexes containing the SMN protein

Abstract

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.

Figure 1

Comparison of the nucleotide sequence of Gemin4 and the corresponding sequence of the clone identified as a ligand of galectin-1. DNA from the putative interacting clone was amplified by PCR and then sequenced. When the sequence of this clone (Query) was subjected to a BLAST search against the GenBank genome database, there was 100% identity with the C-terminal 50 amino acids of Gemin4 (accession no. AF173856).

Figure 2

In vitro binding assay between GST–Gemin4(C50) and galectin-1 and galectin-3. Approximately 1 µg of GST (lanes 2, 5 and 7) or GST–Gemin4(C50) (lanes 3, 6 and 8) was bound to glutathione–Sepharose beads that had been pretreated with bacterial lysate from E.coli cells used for production of the recombinant fusion polypeptides. Bovine galectin-1 (0.5 µg) (A) or recombinant murine galectin-3 (0.5 µg) (B) was incubated with the GST or GST–Gemin4(C50) beads for 2 h at 4°C. After washing, the bound material was eluted and subjected to SDS–PAGE. Galectin-1 and galectin-3 were visualized by immunoblotting. In lanes 1 and 4, 10% of the input test protein is shown. After immunoblotting, one of the membranes was stained with Coomassie blue (C) to ascertain that equal quantities of GST and GST–Gemin4(C50) were bound to the glutathione beads.

Figure 3

Analysis of the polypeptides immunoprecipitated from NE by anti-galectin-1. NE (∼80 µg protein) was incubated for 2 h at 4°C with protein G–Sepharose beads covalently coupled with polyclonal rabbit anti-rat galectin-1. After washing, the bound material was eluted and subjected to SDS–PAGE. Polypeptides (indicated to the right of each panel) in the immunoprecipitates were identified with the following antibodies: affinity purified rabbit polyclonal antibodies against human galectin-1; mouse monoclonal antibody against SMN; mouse monoclonal antibody against Gemin2; rat monoclonal antibody against galectin-3; human autoimmune serum reactive against the Sm epitopes on the core polypeptides of snRNPs; rabbit polyclonal antibodies to HMGs 14/17. In each panel, PI represents material precipitated by preimmune serum; αG1 represents material precipitated by rabbit anti-rat galectin-1; NE represents 50% of the nuclear extract subjected to immunoprecipitation, except the α galectin-1 immunoblot, in which the NE is ∼80% of that used for immunoprecipitation.

Figure 4

Comparison of the effect of addition of recombinant galectin-3 or its N- and C-terminal domains on the splicing activity of nuclear extract. Lane 1, nuclear extract (NE); lanes 2–4, recombinant galectin-3 (rG3) at 3, 6 and 12 µM added to NE; lanes 5–8, ND at 0.6, 1.2, 5 and 10 µM added to NE; lanes 9–12, CD at 3, 10, 28 and 57 µM added to NE. All reactions contained ³²P-labeled MINX pre-mRNA substrate (5000 c.p.m.) and products of the splicing reactions were analyzed by electrophoresis through a 13% polyacrylamide–urea gel, followed by autoradiography. The positions of migration of pre-mRNA substrate, splicing intermediates (exon 1 and lariat–exon 2) and RNA products (lariat and ligated exon 1–exon 2) are highlighted on the right.

Figure 5

Comparison of the effect of addition of recombinant galectin-3 or its N- and C-terminal domains on formation of splicing complexes. All samples contained nuclear extract (NE) and ³²P-labeled MINX pre-mRNA substrate (5000 c.p.m.). The sample in lane 1 was incubated for 5 min in the absence of ATP; the samples in lanes 2–6 were incubated in the presence of ATP. Lane 2, no additions to NE, 0 min; lane 3, no additions to NE, 5 min; lane 4, 12 µM recombinant galectin-3 (rG3), 5 min; lane 5, 10 µM ND, 5 min; lane 6, 10 µM CD, 5 min. Splicing complexes were analyzed by non-denaturing gel electrophoresis and autoradiography. The positions of the H-, A- and B-complexes (22) are highlighted on the right.

Figure 6

The effect of Gemin4(C50) addition on the splicing activity of nuclear extract. Lane 1, nuclear extract (NE); lanes 2 and 3, GST at 10 and 30 µM added to NE; lanes 4 and 5, GST–Gemin4(C50) at 10 and 30 µM added to NE. All reactions contained ³²P-labeled MINX substrate (5000 c.p.m.) and products of the splicing reactions were analyzed by electrophoresis through a 13% polyacrylamide–urea gel, followed by autoradiography. The positions of migration of pre-mRNA substrate, splicing intermediates and RNA product are highlighted on the right.

Figure 7

Schematic diagram summarizing the components of SMN-containing complexes and the intermediates in spliceosome assembly. The known and newly identified polypeptides of SMN complexes are delineated on the left. The steps in active spliceosome formation are shown on the right. The dotted rectangles represent exons and the horizontal line represents the intron on the pre-mRNA. The ATP-independent H- and E-complexes migrate to a position designated as the H-complex in our native gel electrophoretic system. The SMN-containing complexes are illustrated here to supply snRNPs to the H-complex, leading to the formation of ATP-dependent active spliceosomal A-, B- and C-complexes. This delivery may also involve galectin-1 and galectin-3 inasmuch as galectin depletion results in arrest of spliceosome assembly at the H-complex.