Composition of the Survival Motor Neuron (SMN) Complex in Drosophila melanogaster

Abstract

Spinal Muscular Atrophy (SMA) is caused by homozygous mutations in the human survival motor neuron 1 ( SMN1 ) gene. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. SMN is part of an oligomeric complex with core binding partners, collectively called Gemins. Biochemical and cell biological studies demonstrate that certain Gemins are required for proper snRNP assembly and transport. However, the precise functions of most Gemins are unknown. To gain a deeper understanding of the SMN complex in the context of metazoan evolution, we investigated its composition in Drosophila melanogaster . Using transgenic flies that exclusively express Flag-tagged SMN from its native promoter, we previously found that Gemin2, Gemin3, Gemin5, and all nine classical Sm proteins, including Lsm10 and Lsm11, co-purify with SMN. Here, we show that CG2941 is also highly enriched in the pulldown. Reciprocal co-immunoprecipitation reveals that epitope-tagged CG2941 interacts with endogenous SMN in Schneider2 cells. Bioinformatic comparisons show that CG2941 shares sequence and structural similarity with metazoan Gemin4. Additional analysis shows that three other genes ( CG14164 , CG31950 and CG2371 ) are not orthologous to Gemins 6-7-8, respectively, as previously suggested. In D.melanogaster , CG2941 is located within an evolutionarily recent genomic triplication with two other nearly identical paralogous genes ( CG32783 and CG32786 ). RNAi-mediated knockdown of CG2941 and its two close paralogs reveals that Gemin4 is essential for organismal viability.

Intimate functional interactions between TGS1 and the Smn complex revealed by an analysis of the Drosophila eye development

“…Gem5 and Lsm10 were also present in precipitates but fell just outside of our stringent cut-off criteria ( Fig 2D). These findings are in good agreement with recent AP-MS results obtained from Drosophila embryos expressing Smn-FLAG [41]. Collectively, our results provide strong evidence that dTgs1 physically associates with the Drosophila Smn complex in vivo, suggesting a parallel functional interaction ( Fig 2B, 2C and 2D).…”

“…Noteworthy, a physical interaction between TGS1 and SmB has previously been demonstrated in both human and yeast cells [12]. Moreover, consistent with our AP/MS results, Gem5 was the less abundant precipitate among the Gemins that co-purify with Smn-FLAG [41]. Collectively, our findings reveal a strong physical association between dTgs1 and the Smn complex subunits that has not been detected in previous studies on any organism.…”

“…Interestingly, the fly SMN complex contains only four bona fide Gemin proteins (Gem2, Gem3, Gem4/Glos and Gem5/Rig) [41,42,68]. The existence of potential homologs of Gem6, 7 and 8 postulated by Lanfranco et al [42] was not confirmed in a subsequent study [41]. Our AP/MS analyses of embryo extracts showed that the 11 most abundant dTgs1-interacting proteins include Smn and four Gemins (Gem2, Gem3, Gem4a and Gem4b).…”

“…Work in Drosophila has shown that dTgs1 interacts with the Gemin3 subunit of the SMN complex in a two-hybrid assay [37]. Interestingly, the fly SMN complex contains only four bona fide Gemin proteins (Gem2, Gem3, Gem4/Glos and Gem5/Rig) [41,42,68]. The existence of potential homologs of Gem6, 7 and 8 postulated by Lanfranco et al [42] was not confirmed in a subsequent study [41].…”

“…To select bona fide dTgs1-binding partners we applied stringent filtering against a database of non-specific interactors as described by [40]; we selected only proteins IDs that were not found in the negative control and with a score above 50, and ranked them by mean area. Using this criterion, we identified 11 specific dTgs1 interactors that include Smn and four Gemins: Gem2, Gem3 and the protein products of the duplicated genes Gem4a (Glos or CG2941) and Gem4b (CG32786) [41,42] (Fig 2B and 2C). Peptides corresponding to Gem5 were also detected in precipitates, but with an overall score well below our cut-off ( Fig 2C; S1 Table).…”