Evolution of an RNP assembly system: a minimal SMN complex facilitates formation of UsnRNPs in Drosophila melanogaster

Abstract

In vertebrates, assembly of spliceosomal uridine-rich small nuclear ribonucleoproteins (UsnRNPs) is mediated by the SMN complex, a macromolecular entity composed of the proteins SMN and Gemins 2-8. Here we have studied the evolution of this machinery using complete genome assemblies of multiple model organisms. The SMN complex has gained complexity in evolution by a blockwise addition of Gemins onto an ancestral core complex composed of SMN and Gemin2. In contrast to this overall evolutionary trend to more complexity in metazoans, orthologs of most Gemins are missing in dipterans. In accordance with these bioinformatic data a previously undescribed biochemical purification strategy elucidated that the dipteran Drosophila melanogaster contains an SMN complex of remarkable simplicity. Surprisingly, this minimal complex not only mediates the assembly reaction in a manner very similar to its vertebrate counterpart, but also prevents misassembly onto nontarget RNAs. Our data suggest that only a minority of Gemins are required for the assembly reaction per se, whereas others may serve additional functions in the context of UsnRNP biogenesis. The evolution of the SMN complex is an interesting example of how the simplification of a biochemical process contributes to genome compaction.

Fig. 1.

Evolution of the SMN complex. Complete genome assemblies of indicated organisms have been screened for orthologs of human SMN complex components. The presence of the proteins was mapped on a published phylogenetic tree (38). A homolog of Gemin6 was found in the algae O. tauri, but the homology was restricted to the C-terminus and therefore is not shown. Gemin5 orthologs in dipterans are evolving significantly faster than in other organisms. Because this may indicate a change of function, they are shown as squares.

Fig. 2.

TagIt–dSMN binds dGemin2 and Sm proteins. (A) Coimmunoprecipitation of TagIt–Dhh1 with dSMN from Schneider2 cells at increasing salt conditions (lanes 4–6). The coprecipitated dSMN protein was detected by Western blot. Lanes 1–3 show control immunoprecipitations. (B) Extracts from cells expressing TagIt–Rigor mortis (lanes 4–6), TagIt–dSMN (lane 7), or no tagged protein (lanes 1–3) were immunoprecipitated with antibody 7B10. Immunoprecipitates were analyzed by Western blotting with antibodies against dSMN and Rigor mortis, respectively. (C) Extracts from Drosophila Schneider2 cells stably expressing TagIt–dSMN were separated on glycerol gradients and analyzed by Western blotting with 7B10 (Upper) an anti-dGemin2 antibody (Lower). Estimated sedimentation value is indicated. (D) Isolation of TagIt–dSMN from Schneider2 extracts. Proteins were separated by SDS/PAGE under reducing (lanes 1 and 2) and nonreducing (lane 3) conditions and visualized by silver staining. Lane 1 shows a control elution of nontransfected cells. The indicated proteins were identified by mass spectrometry. (E) Immunoblot analyses of TagIt–dSMN (Upper) and TagIt–dGemin2 (Lower) purifications with indicated antibodies. Lanes 1 and 3 show mock controls.

Fig. 3.

dSMN complex contains UsnRNAs and is active in UsnRNP assembly. (A) Northern blot analysis revealed snRNAs U1, U2, U4, and U5 but not Met-tRNA_i in anti-dSMN (lane 3) and anti-dGemin2 (lane 3 and 4) immunoprecipitates from Schneider2 total cell extract. Lane 1 shows the extract before immunoprecipitation, and lane 2 shows a control immunoprecipitation with a normal rabbit serum (NRS). (B) The same set of snRNAs was purified from cytosolic extract of TagIt–dSMN-expressing cells. U4 snRNA was detected after longer exposure of the film. (C) In vitro transcribed U1snRNA, U1ΔSm, and U85scaRNA were incubated with purified TagIt–dSMN complex at the indicated temperatures (lanes 2–4, 6–8, and 10–12) and separated by native gel electrophoresis. Lanes 1, 5, and 9 show the indicated RNAs in the absence of SMN complex; in lanes 4, 8, and 12 monoclonal antibody Y12 was added after the assembly reaction had been completed. The assembled dU1 Sm core domain and the supershift are indicated by arrows.

Fig. 4.

Reconstitution of functional dSMN complex. (A) Recombinant H₆-GST-dGemin2/H₆-dSMN complex was immobilized on glutathione-Sepharose beads and incubated with purified Sm protein heterooligomers as indicated (lanes 9–15). As a specificity control, H₆-GST was immobilized on beads and mock-loaded with Sm proteins (lane 6). After removal of unbound proteins, reconstituted complexes were analyzed by SDS/PAGE. Lanes 1–5 show proteins used for the reconstitution assay. Bands indicated by an asterisk are degradation products or aggregates. In vitro assembly assay of reconstituted dSMN complex with dU1snRNA (B), dU1ΔSm (C), and dU85scaRNA (D) is shown. Radiolabeled RNAs were incubated with His-GST control (lanes 2, 7, and 12), dSMN/dGemin2 dimer (lanes 3, 8, and 13), or dSMN/dGemin2 bound to Sm proteins (lanes 4, 9, and 14), or with free Sm proteins (lanes 5, 10, and 15). Lanes 1, 6, and 11 show the RNA in the absence of protein. Reaction mixtures were separated by native gel electrophoresis, and complexes were visualized by autoradiography. The light upper band seen most prominently in lanes 12 and 14 denotes a conformer of dU85scaRNA.