Sm Proteins Research Articles

Crystal structure of U2 snRNP SF3b components: Hsh49p in complex with Cus1p-binding domain

Spliceosomal proteins Hsh49p and Cus1p are components of SF3b, which together with SF3a, Msl1p/Lea1p, Sm proteins, and U2 snRNA, form U2 snRNP, which plays a crucial role in pre-mRNA splicing. Hsh49p, comprising two RRMs, forms a heterodimer with Cus1p. We determined the crystal structures of Saccharomyces cerevisiae full-length Hsh49p as well as its RRM1 in complex with a minimal binding region of Cus1p (residues 290–368). The structures show that the Cus1 fragment binds to the α-helical surface of Hsh49p RRM1, opposite the four-stranded β-sheet, leaving the canonical RNA-binding surface available to bind RNA. Hsh49p binds the 5′ end region of U2 snRNA via RRM1. Its affinity is increased in complex with Cus1(290-368)p, partly because an extended RNA-binding surface forms across the protein–protein interface. The Hsh49p RRM1–Cus1(290-368)p structure fits well into cryo-EM density of the Bact spliceosome, corroborating the biological relevance of our crystal structure.

The sole LSm complex in Cyanidioschyzon merolae associates with pre-mRNA splicing and mRNA degradation factors.

Proteins of the Sm and Sm-like (LSm) families, referred to collectively as (L)Sm proteins, are found in all three domains of life and are known to promote a variety of RNA processes such as base-pair formation, unwinding, RNA degradation, and RNA stabilization. In eukaryotes, (L)Sm proteins have been studied, inter alia, for their role in pre-mRNA splicing. In many organisms, the LSm proteins form two distinct complexes, one consisting of LSm1–7 that is involved in mRNA degradation in the cytoplasm, and the other consisting of LSm2–8 that binds spliceosomal U6 snRNA in the nucleus. We recently characterized the splicing proteins from the red alga Cyanidioschyzon merolae and found that it has only seven LSm proteins. The identities of CmLSm2–CmLSm7 were unambiguous, but the seventh protein was similar to LSm1 and LSm8. Here, we use in vitro binding measurements, microscopy, and affinity purification-mass spectrometry to demonstrate a canonical splicing function for the C. merolae LSm complex and experimentally validate our bioinformatic predictions of a reduced spliceosome in this organism. Copurification of Pat1 and its associated mRNA degradation proteins with the LSm proteins, along with evidence of a cytoplasmic fraction of CmLSm complexes, argues that this complex is involved in both splicing and cytoplasmic mRNA degradation. Intriguingly, the Pat1 complex also copurifies with all four snRNAs, suggesting the possibility of a spliceosome-associated pre-mRNA degradation complex in the nucleus.

Identification of a small molecule inhibitor that stalls splicing at an early step of spliceosome activation

Small molecule inhibitors of pre-mRNA splicing are important tools for identifying new spliceosome assembly intermediates, allowing a finer dissection of spliceosome dynamics and function. Here, we identified a small molecule that inhibits human pre-mRNA splicing at an intermediate stage during conversion of pre-catalytic spliceosomal B complexes into activated Bact complexes. Characterization of the stalled complexes (designated B028) revealed that U4/U6 snRNP proteins are released during activation before the U6 Lsm and B-specific proteins, and before recruitment and/or stable incorporation of Prp19/CDC5L complex and other Bact complex proteins. The U2/U6 RNA network in B028 complexes differs from that of the Bact complex, consistent with the idea that the catalytic RNA core forms stepwise during the B to Bact transition and is likely stabilized by the Prp19/CDC5L complex and related proteins. Taken together, our data provide new insights into the RNP rearrangements and extensive exchange of proteins that occurs during spliceosome activation.

SM protein Munc18-2 facilitates transition of Syntaxin 11-mediated lipid mixing to complete fusion for T-lymphocyte cytotoxicity

The atypical lipid-anchored Syntaxin 11 (STX11) and its binding partner, the Sec/Munc (SM) protein Munc18-2, facilitate cytolytic granule release by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Patients carrying mutations in these genes develop familial hemophagocytic lymphohistiocytosis, a primary immunodeficiency characterized by impaired lytic granule exocytosis. However, whether a SNARE such as STX11, which lacks a transmembrane domain, can support membrane fusion in vivo is uncertain, as is the precise role of Munc18-2 during lytic granule exocytosis. Here, using a reconstituted "flipped" cell-cell fusion assay, we show that lipid-anchored STX11 and its cognate SNARE proteins mainly support exchange of lipids but not cytoplasmic content between cells, resembling hemifusion. Strikingly, complete fusion is stimulated by addition of wild-type Munc18-2 to the assay, but not of Munc18-2 mutants with abnormal STX11 binding. Our data reveal that Munc18-2 is not just a chaperone of STX11 but also directly contributes to complete membrane merging by promoting SNARE complex assembly. These results further support the concept that SM proteins in general are part of the core fusion machinery. This fusion mechanism likely contributes to other cell-type-specific exocytic processes such as platelet secretion.

A Simulation Based Analysis of the Oligomeric Plasticity of Sm Protein Assemblies

RNA-associated Sm proteins can be found in all three domains of life. In eukarya, Sm proteins are well-studied in connection with their roles mRNA splicing. In bacteria, the Sm protein Hfq acts as an RNA chaperone, playing vital roles in mRNA-sRNA annealing and RNA-based regulatory networks. In archaea, the functional roles of Sm proteins remains an open question. Sm proteins assemble into cyclic oligomers of 5, 6, 7, or 8 subunits, and the assemblies can be either homo- or heteromeric. Bacterial Sm proteins have only been found as homo-hexamers, while eukaryotic Sm proteins typically assemble into hetero-heptamers. Archaeal Sm proteins have been found as homomeric hexamers, heptamers, and octamers. Despite this variation in quaternary structure, all Sm monomers exhibit nearly identical tertiary structures. How can this be? What is the origin of this oligomeric plasticity, if not encoded in the monomer? We have used a systematic array of molecular dynamics simulations to examine the interfaces between Sm subunits, and have developed several quantitative relations that link the results of dimer simulations to the behavior of complete rings. The simulations reveal that Sm oligomers are remarkably flexible. Sm dimers can adopt multiple conformations, and Sm rings are distinctly asymmetric. In particular, the octameric ring of an Sm-like archaeal protein deforms drastically, and dimers of that protein appear to adopt a non-Sm-like tertiary structure. For a dimer of the E. coli Sm protein, our simulations show one monomer twisting nearly fifteen degrees from its position in the crystal structure. The surprising flexibility of Sm oligomers may be related to the dynamical effects of RNA binding and we are currently investigating these effects in a variety of Sm systems.

HOPS catalyzes the interdependent assembly of each vacuolar SNARE into a SNARE complex.

Rab GTPases, their effectors, SNAREs of the R, Qa, Qb, and Qc families, and SM SNARE-binding proteins catalyze intracellular membrane fusion. At the vacuole/lysosome, they are integrated by the homotypic fusion and vacuole protein sorting (HOPS) complex. Two HOPS subunits bind vacuolar Rabs for tethering, another binds the Qc SNARE, and a fourth HOPS subunit, an SM protein, has conserved grooves that bind R- and Qa-SNARE domains. Spontaneous quaternary SNARE complex assembly is very slow. We report an assay of SNARE complex assembly that does not rely on fusion and for which tethering does not coenrich the four SNAREs. HOPS is required in this assay for rapid SNARE complex assembly. Optimal assembly needs HOPS, lipid membranes to which the R- or Qa-SNARE and Ypt7:GTP are integrally bound, and each of the other three SNAREs. Each SNARE assembles into this complex relying on the others, suggesting four-SNARE complex assembly rather than direct binding of each to HOPS. SNAREs can be disassociated by Sec 17/Sec 18/ATP, completing a catalyzed cycle of SNARE assembly and disassembly.

Navigation through the Plasma Membrane Molecular Landscape Shapes Random Organelle Movement

SummaryEukaryotic plasma membrane organization theory has long been controversial, in part due to a dearth of suitably high-resolution techniques to probe molecular architecture in situ and integrate information from diverse data streams [1]. Notably, clustered patterning of membrane proteins is a commonly conserved feature across diverse protein families (reviewed in [2]), including the SNAREs [3], SM proteins [4, 5], ion channels [6, 7], and receptors (e.g., [8]). Much effort has gone into analyzing the behavior of secretory organelles [9, 10, 11, 12, 13], and understanding the relationship between the membrane and proximal organelles [4, 5, 12, 14] is an essential goal for cell biology as broad concepts or rules may be established. Here we explore the generally accepted model that vesicles at the plasmalemma are guided by cytoskeletal tracks to specific sites on the membrane that have clustered molecular machinery for secretion [15], organized in part by the local lipid composition [16]. To increase our understanding of these fundamental processes, we integrated nanoscopy and spectroscopy of the secretory machinery with organelle tracking data in a mathematical model, iterating with knockdown cell models. We find that repeated routes followed by successive vesicles, the re-use of similar fusion sites, and the apparently distinct vesicle “pools” are all fashioned by the Brownian behavior of organelles overlaid on navigation between non-reactive secretory protein molecular depots patterned at the plasma membrane.

Clinical & immunological characteristics in systemic lupus erythematosus patients.

Background & objectives:Systemic lupus erythematosus (SLE) is a chronic autoimmune disease which affects females more than males. Gender affects the manifestations of SLE and men with lupus show more severe symptoms and worse prognosis. This study was aimed to compare clinical and immunological features in female and male lupus patients in Iran.Methods:Demographic, clinical and laboratory data from 78 women and 20 men with lupus were collected. Autoantibodies (against nRNP, Sm, SSA, SSB, Ro-52, CENP, Jo-1, Scl-70, nucleosome, anti-dsDNA, histone and Rib-p protein) were determined using immunoblotting technique.Results:Men with lupus had less anti-SSA (21.1 vs 48.1%) and anti-Ro52 (10.5 vs 44.3%) antibodies when compared to women and none of the male patients had anti-SSB antibodies. Kidney damage was more frequent in men (68.4% in men vs 36.7% in women). In men with kidney involvement, anti-dsDNA increased significantly (84.6 vs 20.0%) in comparison to males without nephritis. Anti-SSA (7.7 vs 50.0%) and anti-nRNP (0.0 vs 33.8%) on the other hand, decreased. Women with renal involvement had no anti-SSB antibodies.Interpretation & conclusions:In male patients, SLE appeared with more severe features, and kidney damage was more frequent in males. The frequency of some autoantibodies was different between females and males. In males with kidney damage anti-dsDNA increased significantly, while anti-SSA and anti-nRNP decreased. Anti-SSB was not detected in males and females with nephritis.

Will the circle be unbroken: specific mutations in the yeast Sm protein ring expose a requirement for assembly factor Brr1, a homolog of Gemin2.

A seven-subunit Sm protein ring assembles around specific U-rich RNA segments of the U1, U2, U4, and U5 snRNPs that direct pre-mRNA splicing. Using human snRNP crystal structures to guide mutagenesis in Saccharomyces cerevisiae, we gained new insights into structure–function relationships of the SmD1 and SmD2 subunits. Of 18 conserved amino acids comprising their RNA-binding sites or intersubunit interfaces, only Arg88 in SmD1 and Arg97 in SmD2 were essential for growth. Tests for genetic interactions with non-Sm splicing factors identified benign mutations of SmD1 (N37A, R88K, R93A) and SmD2 (R49A, N66A, R97K, D99A) that were synthetically lethal with null alleles of U2 snRNP subunits Lea1 and/or Msl1. Tests of 264 pairwise combinations of SmD1 and SmD2 alleles with each other and with a collection of SmG, SmE, SmF, SmB, and SmD3 alleles revealed 92 instances of inter-Sm synthetic lethality. We leveraged the Sm mutant collection to illuminate the function of the yeast Sm assembly factor Brr1 and its relationship to the metazoan Sm assembly factor Gemin2. Mutations in the adjacent SmE (K83A), SmF (K32A, F33A, R74K), SmD2 (R49A, N66A, E74A, R97K, D99A), and SmD1 (E18A, N37A) subunits—but none in the SmG, SmD3, and SmB subunits—were synthetically lethal with brr1Δ. Using complementation of brr1Δ lethality in two Sm mutant backgrounds as an in vivo assay of Brr1 activity, we identified as essential an N-terminal segment of Brr1 (amino acids 24–47) corresponding to the Gemin2 α1 helix that interacts with SmF and a Brr1 C-terminal peptide (336QKDLIE341) that, in Gemin2, interacts with SmD2.

Structural basis for the recognition of spliceosomal SmN/B/B' proteins by the RBM5 OCRE domain in splicing regulation.

The multi-domain splicing factor RBM5 regulates the balance between antagonistic isoforms of the apoptosis-control genes FAS/CD95, Caspase-2 and AID. An OCRE (OCtamer REpeat of aromatic residues) domain found in RBM5 is important for alternative splicing regulation and mediates interactions with components of the U4/U6.U5 tri-snRNP. We show that the RBM5 OCRE domain adopts a unique β-sheet fold. NMR and biochemical experiments demonstrate that the OCRE domain directly binds to the proline-rich C-terminal tail of the essential snRNP core proteins SmN/B/B'. The NMR structure of an OCRE-SmN peptide complex reveals a specific recognition of poly-proline helical motifs in SmN/B/B'. Mutation of conserved aromatic residues impairs binding to the Sm proteins in vitro and compromises RBM5-mediated alternative splicing regulation of FAS/CD95. Thus, RBM5 OCRE represents a poly-proline recognition domain that mediates critical interactions with the C-terminal tail of the spliceosomal SmN/B/B' proteins in FAS/CD95 alternative splicing regulation.

A Novel Mutation in Roifman Syndrome Redefines the Boundaries of the Sm Protein-Binding Site

Roifman syndrome is an association of humoral immunodeficiency, growth retardation, spondyloepiphyseal dysplasia, developmental delay, retinal dystrophy, and unique dysmorphism. Compound heterozygote mutations in the RNU4ATAC gene, an essential component of the minor spliceosome, were found to be the culprit for this disorder. Here we report a novel mutation in the RNU4ATAC gene, involving position 116. This mutation redefines the Sm protein binding site of this gene.Statement of novelty: We report here a patient with a novel mutation in the Sm protein-binding site that redefined its boundaries to include position 116.

Interactions between Giardia duodenalis Sm proteins and their association with spliceosomal snRNAs.

Giardia duodenalis is a parasite that colonises the intestines of humans and other vertebrates, causing diarrhoea and poor nutrient absorption. G. duodenalis is sometimes considered an early diverging eukaryote, and its genome exhibits simplified molecular machinery for many cellular processes, which makes it an interesting model to study. The spliceosome, one of the most complex molecular machines in the eukaryotic cell, is responsible for intron excision and exon splicing. Just over a decade ago, it was believed that the G. duodenalis genome did not contain introns or undergo splicing. Research now shows that this speculation was incorrect and that uncommon mechanisms, such as trans-splicing from different genes, occur. In silico studies of the parasite suggest the possibility of a simplified spliceosome and spliceosomal small nuclear RNA (snRNA) candidates; however, none of these components have been identified in vivo. Here, we developed a strategy to study the in vivo expression, interactions and localisation of these spliceosome components in G. duodenalis. Haemagglutinin (HA)-tagged SmB and SmD3 proteins, which form part of the spliceosome core, were overexpressed in the parasite. Immunoprecipitation with anti-HA revealed that the SmD3 protein is associated with the proteins SmB, SmD1, SmD2, SmE and SmF in vivo. In addition, the U1, U2 and U4 snRNA candidates reported previously were found in the protein complex, suggesting that these molecules are spliceosomal snRNAs of G. duodenalis and they contained a 2,2,7-trimethylguanosine modification at their 5' end. Our data indicate that the actively expressed spliceosome in G. duodenalis is similar to that of highly evolved protists and higher animals.

Structural basis for snRNA recognition by the double-WD40 repeat domain of Gemin5.

Assembly of the spliceosomal small nuclear ribonucleoparticle (snRNP) core requires the participation of the multisubunit SMN (survival of motor neuron) complex, which contains SMN and several Gemin proteins. The SMN and Gemin2 subunits directly bind Sm proteins, and Gemin5 is required for snRNP biogenesis and has been implicated in snRNA recognition. The RNA sequence required for snRNP assembly includes the Sm site and an adjacent 3' stem-loop, but a precise understanding of Gemin5's RNA-binding specificity is lacking. Here we show that the N-terminal half of Gemin5, which is composed of two juxtaposed seven-bladed WD40 repeat domains, recognizes the Sm site. The tandem WD40 repeat domains are rigidly held together to form a contiguous RNA-binding surface. RNA-contacting residues are located mostly on loops between β strands on the apical surface of the WD40 domains. Structural and biochemical analyses show that base-stacking interactions involving four aromatic residues and hydrogen bonding by a pair of arginines are crucial for specific recognition of the Sm sequence. We also show that an adenine immediately 5' to the Sm site is required for efficient binding and that Gemin5 can bind short RNA oligos in an alternative mode. Our results provide mechanistic understandings of Gemin5's snRNA-binding specificity as well as valuable insights into the molecular mechanism of RNA binding by WD40 repeat proteins in general.

Distinct roles of the two VPS33 proteins in the endolysosomal system in Caenorhabditis elegans

Sec1/Munc-18 (SM) family proteins are essential regulators in intracellular transport in eukaryotic cells. The SM protein Vps33 functions as a core subunit of two tethering complexes, class C core vacuole/endosome tethering (CORVET) and homotypic fusion and vacuole protein sorting (HOPS) in the endocytic pathway in yeast. Metazoan cells possess two Vps33 proteins, VPS33A and VPS33B, but their precise roles remain unknown. Here, we present a comparative analysis of Caenorhabditis elegans null mutants for these proteins. We found that the vps-33.1 (VPS33A) mutants exhibited severe defects in both endocytic function and endolysosomal biogenesis in scavenger cells. Furthermore, vps-33.1 mutations caused endocytosis defects in other tissues, and the loss of maternal and zygotic VPS-33.1 resulted in embryonic lethality. By contrast, vps-33.2 mutants were viable but sterile, with terminally arrested spermatocytes. The spermatogenesis phenotype suggests that VPS33.2 is involved in the formation of a sperm-specific organelle. The endocytosis defect in the vps-33.1 mutant was not restored by the expression of VPS-33.2, which indicates that these proteins have nonredundant functions. Together, our data suggest that VPS-33.1 shares most of the general functions of yeast Vps33 in terms of tethering complexes in the endolysosomal system, whereas VPS-33.2 has tissue/organelle specific functions in C. elegans.

C-terminal domain of the RNA chaperone Hfq drives sRNA competition and release of target RNA

The bacterial Sm protein and RNA chaperone Hfq stabilizes small noncoding RNAs (sRNAs) and facilitates their annealing to mRNA targets involved in stress tolerance and virulence. Although an arginine patch on the Sm core is needed for Hfq's RNA chaperone activity, the function of Hfq's intrinsically disordered C-terminal domain (CTD) has remained unclear. Here, we use stopped flow spectroscopy to show that the CTD of Escherichia coli Hfq is not needed to accelerate RNA base pairing but is required for the release of dsRNA. The Hfq CTD also mediates competition between sRNAs, offering a kinetic advantage to sRNAs that contact both the proximal and distal faces of the Hfq hexamer. The change in sRNA hierarchy caused by deletion of the Hfq CTD in E. coli alters the sRNA accumulation and the kinetics of sRNA regulation in vivo. We propose that the Hfq CTD displaces sRNAs and annealed sRNA⋅mRNA complexes from the Sm core, enabling Hfq to chaperone sRNA-mRNA interactions and rapidly cycle between competing targets in the cell.

Effects of co-overexpression of secretion helper factors on the secretion of a HSA fusion protein (IL2-HSA) in pichia pastoris.

Pichia pastoris is generally considered as an expression host for heterologous proteins with the coding gene under control of the alcohol oxidase 1 (AOX1) promoter. The secretion of heterologous proteins in P. pastoris can be potentially affected by many factors. Based on our previous results, the secretion levels of human albumin (HSA) fusion protein IL2-HSA were only around 500 mg/L or less in fermentor cultures, which decreased more than 50% compared with that of HSA (>1 g/L). In this study, we selected five potential secretion helper factors, in which Ero1, Pdi1 and Kar2 were involved in protein folding and Sec1 and Sly1 were involved in vesicle trafficking. We evaluated the possible effects of individual overexpression of these secretion helper factors on the secretion of IL2-HSA in P. pastoris. Constitutive overexpression of the five selected secretion factors did not have an obvious negative effect on cell growth of the IL2-HSA secreting strain. Individual co-overexpression of Ero1, Kar2, Pdi1, Sec1 and Sly1 improved the secretion level of IL2-HSA to ~2.3-, 1.9-, 2.2-, 2.5- and 1.9-fold that in the control strain respectively in shake flasks. We evaluated the changes in mRNA and protein levels of the intracellular IL2-HSA, as well as the secretion helper factor genes in the co-overexpressing strains. Our results indicated that manipulating the expression level of ER resident protein Pdi1, Ero1, Kar2 and SM protein Sec1 and Sly1 could improve the secretion level of IL2-HSA fusion protein in P. pastoris, which provided new candidates for combinatorial engineering in future study. Copyright © 2016 John Wiley & Sons, Ltd.

Characterization of RNA-binding properties of the archaeal Hfq-like protein from Methanococcus jannaschii

The Sm and Sm-like proteins are widely distributed among bacteria, archaea and eukarya. They participate in many processes related to RNA-processing and regulation of gene expression. While the function of the bacterial Lsm protein Hfq and eukaryotic Sm/Lsm proteins is rather well studied, the role of Lsm proteins in Archaea is investigated poorly. In this work, the RNA-binding ability of an archaeal Hfq-like protein from Methanococcus jannaschii has been studied by X-ray crystallography, anisotropy fluorescence and surface plasmon resonance. It has been found that MjaHfq preserves the proximal RNA-binding site that usually recognizes uridine-rich sequences. Distal adenine-binding and lateral RNA-binding sites show considerable structural changes as compared to bacterial Hfq. MjaHfq did not bind mononucleotides at these sites and would not recognize single-stranded RNA as its bacterial homologues. Nevertheless, MjaHfq possesses affinity to poly(A) RNA that seems to bind at the unstructured positive-charged N-terminal tail of the protein.

Mutagenic Analysis of the C-Terminal Extension of Lsm1.

The Sm-like proteins (also known as Lsm proteins) are ubiquitous in nature and exist as hexa or heptameric RNA binding complexes. They are characterized by the presence of the Sm-domain. The Lsm1 through Lsm7 proteins are highly conserved in eukaryotes and they form a hetero-octameric complex together with the protein Pat1. The Lsm1-7-Pat1 complex plays a key role in mRNA decapping and 3’-end protection and therefore is required for normal mRNA decay rates in vivo. Lsm1 is a key subunit that is critical for the unique RNA binding properties of this complex. We showed earlier that unlike most Sm-like proteins, Lsm1 uniquely requires both its Sm domain and its C-terminal extension to contribute to the function of the Lsm1-7-Pat1 complex and that the C-terminal segment can associate with the rest of the complex and support the function even in trans. The studies presented here identify a set of residues at the very C-terminal end of Lsm1 to be functionally important and suggest that these residues support the function of the Lsm1-7-Pat1 complex by facilitating RNA binding either directly or indirectly.

Structure-function analysis and genetic interactions of the SmG, SmE, and SmF subunits of the yeast Sm protein ring.

A seven-subunit Sm protein ring forms a core scaffold of the U1, U2, U4, and U5 snRNPs that direct pre-mRNA splicing. Using human snRNP structures to guide mutagenesis in Saccharomyces cerevisiae, we gained new insights into structure–function relationships of the SmG, SmE, and SmF subunits. An alanine scan of 19 conserved amino acids of these three proteins, comprising the Sm RNA binding sites or inter-subunit interfaces, revealed that, with the exception of Arg74 in SmF, none are essential for yeast growth. Yet, for SmG, SmE, and SmF, as for many components of the yeast spliceosome, the effects of perturbing protein–RNA and protein–protein interactions are masked by built-in functional redundancies of the splicing machine. For example, tests for genetic interactions with non-Sm splicing factors showed that many benign mutations of SmG, SmE, and SmF (and of SmB and SmD3) were synthetically lethal with null alleles of U2 snRNP subunits Lea1 and Msl1. Tests of pairwise combinations of SmG, SmE, SmF, SmB, and SmD3 alleles highlighted the inherent redundancies within the Sm ring, whereby simultaneous mutations of the RNA binding sites of any two of the Sm subunits are lethal. Our results suggest that six intact RNA binding sites in the Sm ring suffice for function but five sites may not.

Disruption of snRNP biogenesis factors Tgs1 and pICln induces phenotypes that mirror aspects of SMN-Gemins complex perturbation in Drosophila, providing new insights into spinal muscular atrophy

The neuromuscular disorder, spinal muscular atrophy (SMA), results from insufficient levels of the survival motor neuron (SMN) protein. Together with Gemins 2–8 and Unrip, SMN forms the large macromolecular SMN-Gemins complex, which is known to be indispensable for chaperoning the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). It remains unclear whether disruption of this function is responsible for the selective neuromuscular degeneration in SMA. In the present study, we first show that loss of wmd, the Drosophila Unrip orthologue, has a negative impact on the motor system. However, due to lack of a functional relationship between wmd/Unrip and Gemin3, it is likely that Unrip joined the SMN-Gemins complex only recently in evolution. Second, we uncover that disruption of either Tgs1 or pICln, two cardinal players in snRNP biogenesis, results in viability and motor phenotypes that closely resemble those previously uncovered on loss of the constituent members of the SMN-Gemins complex. Interestingly, overexpression of both factors leads to motor dysfunction in Drosophila, a situation analogous to that of Gemin2. Toxicity is conserved in the yeast S. pombe where pICln overexpression induces a surplus of Sm proteins in the cytoplasm, indicating that a block in snRNP biogenesis is partly responsible for this phenotype. Importantly, we show a strong functional relationship and a physical interaction between Gemin3 and either Tgs1 or pICln. We propose that snRNP biogenesis is the pathway connecting the SMN-Gemins complex to a functional neuromuscular system, and its disturbance most likely leads to the motor dysfunction that is typical in SMA.