Interaction between the small-nuclear-RNA cap hypermethylase and the spinal muscular atrophy protein, survival of motor neuron

Abstract

The biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) requires the cytoplasmic assembly of the Sm-core complex, followed by the hypermethylation of the small nuclear RNA (snRNA) 5' cap. Both the Sm-core complex and the snRNA trimethylguanosine cap are required for the efficient nuclear import of snRNPs. Here, we show that trimethylguanosine synthase 1 (TGS1), the human homologue of the yeast snRNA cap hypermethylase, interacts directly with the survival of motor neuron (SMN) protein. Both proteins are similarly distributed, localizing in the cytoplasm and in nuclear Cajal bodies. The interaction between TGS1 and SMN is disrupted by a mutation in SMN that mimics the predominant isoform of the protein that is expressed in patients with the neurodegenerative disease, spinal muscular atrophy. These data indicate that, in addition to its function in cytoplasmic Sm-core assembly, the SMN protein also functions in the recruitment of the snRNA cap hypermethylase.

Figure 1

Interaction of human trimethylguanosine synthase 1 with the carboxyl tail of SmB and the survival of motor neuron protein. (A) The glutathione-S-transferase (GST) fusion proteins indicated were incubated with [³⁵S]-labelled human trimethylguanosine synthase 1 (TGS1), which was prepared by in vitro transcription and translation using rabbit reticulocyte lysate. Bound proteins were analysed by SDS–polyacrylamide gel electrophoresis (SDS–PAGE). 'Input' indicates aliquots of radioactive proteins that corresponded to 10% of that used in each binding reaction. (B) Anti-TGS1 recognizes endogenous TGS1 as well as the green fluorescent protein (GFP)–TGS1 fusion protein and a fusion protein consisting of the carboxyl terminal of TGS1 tagged with GFP (GFP–Cter). The GFP fusion proteins indicated were transfected into HeLa cells and the extracts were separated by SDS–PAGE. Western blotting analysis was performed using anti-TGS1 antibodies. (C) The survival of motor neuron (SMN) protein was immunoprecipitated from a cytoplasmic extract using anti-TGS1 antibodies. Immuno-precipitation was performed using HeLa S100 extract and anti-TGS1 or preimmune serum (PI), and the proteins were immunoblotted with anti-TGS1 (upper panel) and anti-SMN (lower panel) antibodies. 'Tot' indicates 10% of the input. (D) TGS1 was immunoprecipitated from a cytoplasmic extract using anti-SMN antibodies. Immunoprecipitation was performed using HeLa cytoplasmic S100 extract and anti-SMN or PI, and the proteins were immunoblotted with anti-TGS1 (upper panel) and anti-SMN (lower panel) antibodies.

Figure 2

Direct binding of human trimethylguanosine synthase 1 to the survival of motor neuron protein. (A) Binding assays using recombinant glutathione-S-transferase (GST)–trimethylguanosine synthase 1 (TGS1) and fusions of GST to the amino and carboxyl termini of TGS1 (GST–Nter and GST–Cter, respectively) with [³⁵S]-labelled survival of motor neuron (SMN) that was in vitro translated using rabbit reticulocyte lysate. Bound proteins were analysed by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and autoradiography. (B) The GST fusion proteins indicated were tested for binding with [³⁵S]-labelled SmB protein and treated as described for (A). (C) Binding of the GST–TGS1 protein to in vitro translated [³⁵S]-labelled SMN using an S30 bacterial extract. Proteins bound to GST beads were analysed by SDS–PAGE and autoradiography. (D) The C-terminal region of SMN mediates the interaction with TGS1. The indicated [³⁵S]-labelled SMN proteins were mixed with the GST–TGS1 or GST proteins, and bound proteins were analysed by SDS–PAGE and autoradiography. Note that the amount of [³⁵S]-labelled SMN(Ex4567) input is lower compared with the other [³⁵S]-labelled SMN domains. 'Input' indicates aliquots of radioactive proteins that corresponded to 10% of that used in each binding reaction.

Figure 3

Colocalization of the human trimethylguanosine synthase 1 and survival of motor neuron proteins. HeLa cells carrying green fluorescent protein (GFP)–trimethylguanosine-synthase-1 (TGS1; upper row), GFP–survival-of-motor-neuron (SMN; middle row) or GFP–SMNΔN27 (lower row) constructs were examined by immunofluorescence microscopy using anti-SMN or anti-TGS1 antibodies. Colocalization of the GFP fusion proteins (green) and SMN or TGS1 (red) is indicated by the yellow colour in the combined panels. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI; blue).

Figure 4

Snurportin 1 does not form a complex with human trimethylguanosine synthase 1 in vivo. (A) HeLa cells were transiently transfected with green fluorescent protein (GFP)–trimethylguanosine-synthase-1 (TGS1) and total cell lysates were immunoprecipitated with anti-GFP antibodies. The immunoprecipitates were then analysed by western blotting using the antibodies indicated. Normal mouse serum (NMS) was used as a non-immune control. (B) HeLa cells were transfected with GFP–snurportin 1 (SPN1) and immunoprecipitation experiments were performed either with NMS or with anti-GFP antibodies. Blots were probed with the indicated antibodies. (C) Cytoplasmic S100 extract was immunoprecipitated with anti-TGS1, anti-survival-of-motor-neuron (SMN) and preimmune serum (PI). Immunoprecipitates were analysed by SDS–polyacrylamide gel electrophoresis and immunoblotted with anti-TGS1 (upper panel) and anti-SPN1 (lower panel) antibodies. 'Tot' indicates 2% of the input. 'Input' indicates aliquots of radioactive proteins that corresponded to 2% of that used in each binding reaction.

Figure 5

Disruption of the interaction between the SMNΔEx7 mutant and human trimethylguanosine synthase 1. (A) In vitro translated [³⁵S]-labelled SMN, [³⁵S]-labelled SMNΔN27 and [³⁵S]-labelled SMNΔEx7 were incubated with recombinant glutathione-S-transferase (GST)–trimethylguanosine-synthase-1 (TGS1) or GST. Bound proteins were analysed by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and autoradiography. (B) Hela cells were transfected with the ZZ-tagged SMN alleles indicated and immunoprecipitation experiments were performed with either normal rabbit serum (NRS) or rabbit IgG and protein-A–sepharose beads. Immunocomplexes were then separated by SDS–PAGE and immunoblotted with anti-TGS1 (upper panel) or anti-SMN (lower panel) antibodies. SMN, survival of motor neuron protein. 'Input' indicates aliquots of radioactive proteins that corresponded to 10% of that used in each binding reaction.

Figure 6

Model of TGS1 and SMN interactions in cytoplasmic snRNP biogenesis. Filled red circles represent the carboxy-terminal extensions of SmB, SmD1 and SmD3 proteins. The defect of the SMNΔN27 mutant is also indicated. See text for more details. m₃G, 2,2,7-trimethylguanosine cap; m⁷G, 7-methylguanosine cap; PHAX/CBC; SMNc, survival of motor neuron complex; snRNP, small nuclear ribonucleoprotein; SPN1, snurportin 1; TGS1, trimethylguanosine synthase 1.