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Protein oligomerization is one mechanism by which homogenous solutions can separate into distinct liquid phases, enabling assembly of membraneless organelles. Survival Motor Neuron (SMN) is the eponymous component of a large macromolecular complex that chaperones biogenesis of eukaryotic ribonucleoproteins and localizes to distinct membraneless organelles in both the nucleus and cytoplasm. SMN forms the oligomeric core of this complex, and missense mutations within its YG box domain are known to cause Spinal Muscular Atrophy (SMA). The SMN YG box utilizes a unique variant of the glycine zipper motif to form dimers, but the mechanism of higher-order oligomerization remains unknown. Here, we use a combination of molecular genetic, phylogenetic, biophysical, biochemical and computational approaches to show that formation of higher-order SMN oligomers depends on a set of YG box residues that are not involved in dimerization. Mutation of key residues within this new structural motif restricts assembly of SMN to dimers and causes locomotor dysfunction and viability defects in animal models.

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). RNAi-mediated knockdown of CG2941 and its two close paralogs reveals that Gemin4 is essential for organismal viability.

SMN protein levels inversely correlate with the severity of spinal muscular atrophy. The SCFSlmbE3 ligase complex interacts with a degron embedded within the C-terminal self-oligomerization domain of SMN. The findings elucidate a model whereby accessibility of the SMN degron is regulated by self-multimerization., Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1. Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor neuron (SMN) isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type (WT) SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers.

Spinal muscular atrophy (SMA) is the leading genetic cause of death in young children, arising from homozygous deletion or mutation of theSMN1gene. SMN protein expressed from a paralogous gene,SMN2, is the primary genetic modifier of SMA; small changes in overall SMN levels cause dramatic changes in disease severity. Thus, deeper insight into mechanisms that regulate SMN protein stability should lead to better therapeutic outcomes. Here, we show that SMA patient-derived missense mutations in theDrosophilaSMN Tudor domain exhibit a pronounced temperature sensitivity that affects organismal viability, larval locomotor function, and adult longevity. These disease-related phenotypes are domain-specific and result from decreased SMN stability at elevated temperature. This system was utilized to manipulate SMN levels during various stages ofDrosophiladevelopment. Due to a large maternal contribution of mRNA and protein,Smnis not expressed zygotically during embryogenesis. Interestingly, we find that only baseline levels of SMN are required during larval stages, whereas high levels of protein are required during pupation. This previously uncharacterized period of elevated SMN expression, during which the majority of adult tissues are formed and differentiated, could be an important and translationally relevant developmental stage in which to study SMN function. Altogether, these findings illustrate a novelin vivorole for the SMN Tudor domain in maintaining SMN homeostasis and highlight the necessity for high SMN levels at critical developmental timepoints that is conserved fromDrosophilato humans.

Spinal muscular atrophy (SMA) is the leading genetic cause of death in young children, arising from homozygous deletion or mutation of the survival motor neuron 1 (SMN1) gene. SMN protein expressed from a paralogous gene, SMN2, is the primary genetic modifier of SMA; small changes in overall SMN levels cause dramatic changes in disease severity. Thus, deeper insight into mechanisms that regulate SMN protein stability should lead to better therapeutic outcomes. Here, we show that SMA patient-derived missense mutations in the Drosophila SMN Tudor domain exhibit a pronounced temperature sensitivity that affects organismal viability, larval locomotor function and adult longevity. These disease-related phenotypes are domain specific and result from decreased SMN stability at elevated temperature. This system was utilized to manipulate SMN levels during various stages of Drosophila development. Owing to a large maternal contribution of mRNA and protein, Smn is not expressed zygotically during embryogenesis. Interestingly, we find that only baseline levels of SMN are required during larval stages, whereas high levels of the protein are required during pupation. This previously uncharacterized period of elevated SMN expression, during which the majority of adult tissues are formed and differentiated, could be an important and translationally relevant developmental stage in which to study SMN function. Taken together, these findings illustrate a novel in vivo role for the SMN Tudor domain in maintaining SMN homeostasis and highlight the necessity for high SMN levels at crucial developmental time points that are conserved from Drosophila to humans., Summary: Using animal models of spinal muscular atrophy, we describe a novel disease mechanism caused by temperature-sensitive protein unfolding/instability of the Tudor domain of SMN.

Splicing factor SRSF1 is upregulated in human breast tumors, and its overexpression promotes transformation of mammary cells. Using RNA-seq, we identified SRSF1-regulated alternative splicing (AS) targets in organotypic three-dimensional MCF-10A cell cultures that mimic a context relevant to breast cancer. We identified and validated hundreds of endogenous SRSF1-regulated AS events. De-novo discovery of the SRSF1 binding motif reconciled discrepancies in previous motif analyses. Using a Bayesian model, we determined positional effects of SRSF1 binding on cassette exons: binding close to the 5′ splice site generally promoted exon inclusion, whereas binding near the 3′ splice site promoted either exon skipping or inclusion. Finally, we identified SRSF1-regulated AS events deregulated in human tumors; overexpressing one such isoform, exon-9-included CASC4, increased acinar size and proliferation, and decreased apoptosis, partially recapitulating SRSF1's oncogenic effects. Thus, we uncovered SRSF1 positive and negative regulatory mechanisms, and oncogenic AS events that represent potential targets for therapeutics development.

Survival Motor Neuron (SMN) protein localizes to both the nucleus and the cytoplasm. Cytoplasmic SMN is diffusely localized in large oligomeric complexes with core member proteins, called Gemins. Biochemical and cell biological studies have demonstrated that the SMN complex is required for the cytoplasmic assembly and nuclear transport of Sm-class ribonucleoproteins (RNPs). Nuclear SMN accumulates with spliceosomal small nuclear (sn)RNPs in Cajal bodies, sub-domains involved in multiple facets of snRNP maturation. Thus, the SMN complex forms stable associations with both nuclear and cytoplasmic snRNPs, and plays a critical role in their biogenesis. In this review, we focus on potential functions of the nuclear SMN complex, with particular emphasis on its role within the Cajal body.

Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1. Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of SMN isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phospho-degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric, and sequestered when SMN forms higher-order multimers.