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 the composition of the SMN complex in Drosophila melanogaster. Using a stable transgenic line that exclusively expresses 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). RNAimediated 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 ( Figure 2C). These findings are in good agreement with recent AP-MS results obtained from Drosophila embryos expressing Smn-FLAG [49]. Collectively, our results provide strong evidence that dTgs1 physically associates with the Drosophila Smn complex in vivo, suggesting a parallel functional interaction.…”

“…Interestingly, the fly SMN complex contains only four bona fide Gemin proteins (Gem2, Gem3, Gem4/Glos and Gem5/Rig) [49,50,74]. The existence of potential homologs of Gem6, 7 and 8 was postulated by [50] but was not confirmed in a subsequent study [49]. Our AP/MS analyses of embryo extracts showed that Smn strongly co-precipitates with GFP-dTgs1.…”

“…Work in Drosophila has shown that that dTgs1 interacts with the Gemin3 subunit of the SMN complex in a two-hybrid assay (Borg et al, 2016). Interestingly, the fly SMN complex contains only four Gemin proteins (Gem2, Gem3, Gem4/Glos and Gem5/Rig) and lacks Gem6, 7 and 8, whose coding genes are not present in the Drosophila genome (Cauchi et al, 2010;Matera et al, 2019). Our AP/MS analyses of embryo extracts showed that Smn strongly co-precipitates with GFP-dTgs1.…”

“…shown that that dTgs1 interacts with the Gemin3 subunit of the SMN complex in a twohybrid assay [37]. Interestingly, the fly SMN complex contains only four bona fide Gemin proteins (Gem2, Gem3, Gem4/Glos and Gem5/Rig) [49,50,74]. The existence of potential homologs of Gem6, 7 and 8 was postulated by [50] but was not confirmed in a subsequent study [49].…”

A novel dimerization module in Gemin5 is critical for protein recruitment and translation control

“…Generally, RBPs comprise RNA-binding domains (RBD) and protein-protein interaction modules (Ellisdon et al, 2012;Katibah et al, 2013;Lunde et al, 2007;Yang et al, 2012), such that the combination of distinct domains provides multiple features to these factors. Gemin5 is a predominantly cytoplasmic RBP that forms part of the survival of motor neuron (SMN) complex in metazoan organisms (Battle et al, 2006;Matera et al, 2019). This multi-protein complex plays a critical role in the biogenesis of small nuclear ribonucleoproteins (snRNPs) (Lau et al, 2009), the components of the splicing machinery.…”