Gemin4. A novel component of the SMN complex that is found in both gems and nucleoli

The survival of motor neurons (SMN) protein, the product of the neurodegenerative disease spinal muscular atrophy (SMA) gene, is localized both in the cytoplasm and in discrete nuclear bodies called gems. In both compartments SMN is part of a large complex that contains several proteins including Gemin2 (formerly SIP1) and the DEAD box protein Gemin3. In the cytoplasm, the SMN complex is associated with snRNP Sm core proteins and plays a critical role in spliceosomal snRNP assembly. In the nucleus, SMN is required for pre-mRNA splicing by serving in the regeneration of spliceosomes. These functions are likely impaired in cells of SMA patients because they have reduced levels of functional SMN. Here, we report the identification by nanoelectrospray mass spectrometry of a novel component of the SMN complex that we name Gemin4. Gemin4 is associated in vivo with the SMN complex through a direct interaction with Gemin3. The tight interaction of Gemin4 with Gemin3 suggests that it could serve as a cofactor of this DEAD box protein. Gemin4 also interacts directly with several of the Sm core proteins. Monoclonal antibodies against Gemin4 efficiently immunoprecipitate the spliceosomal U snRNAs U1 and U5 from Xenopus oocytes cytoplasm. Immunolocalization experiments show that Gemin4 is colocalized with SMN in the cytoplasm and in gems. Interestingly, Gemin4 is also detected in the nucleoli, suggesting that the SMN complex may also function in preribosomal RNA processing or ribosome assembly.     

Deletion of murine SMN exon 7 directed to skeletal muscle leads to severe muscular dystrophy

Spinal muscular atrophy (SMA) is characterized by degeneration of motor neurons of the spinal cord associated with muscle paralysis and caused by mutations of the survival motor neuron gene (SMN). To determine whether SMN gene defect in skeletal muscle might have a role in SMA pathogenesis, deletion of murine SMN exon 7, the most frequent mutation found in SMA, has been restricted to skeletal muscle by using the Cre-loxP system. Mutant mice display ongoing muscle necrosis with a dystrophic phenotype leading to muscle paralysis and death. The dystrophic phenotype is associated with elevated levels of creatine kinase activity, Evans blue dye uptake into muscle fibers, reduced amount of dystrophin and upregulation of utrophin expression suggesting a destabilization of the sarcolemma components. The mutant mice will be a valuable model for elucidating the underlying mechanism. Moreover, our results suggest a primary involvement of skeletal muscle in human SMA, which may contribute to motor defect in addition to muscle denervation caused by the motor neuron degeneration. These data may have important implications for the development of therapeutic strategies in SMA.     

Gemin5 Binds to the Survival Motor Neuron mRNA to Regulate SMN Expression

Reduced expression of SMN causes spinal muscular atrophy, a severe neurodegenerative disease. Despite the importance of maintaining SMN levels, relatively little is known about the mechanisms by which SMN levels are regulated. We show here that Gemin5, the snRNA-binding protein of the SMN complex, binds directly to the SMN mRNA and regulates SMN expression. Gemin5 binds with high specificity, both in vitro and in vivo, to sequence and structural elements in the SMN mRNA 3′-untranslated region that are reminiscent of the snRNP code to which Gemin5 binds on snRNAs. Reduction of Gemin5 redistributes the SMN mRNA from heavy polysomes to lighter polysomes and monosomes, suggesting that Gemin5 functions as an activator of SMN translation. SMN protein is not stoichiometrically present on the SMN mRNA with Gemin5, but the mRNA-binding activity of Gemin5 is dependent on SMN levels, providing a feedback mechanism for SMN to regulate its own expression via Gemin5. This work both reveals a new autoregulatory pathway governing SMN expression, and identifies a new mechanism through which SMN can modulate specific mRNA expression via Gemin5.     

Gemin3: A novel DEAD box protein that interacts with SMN, the spinal muscular atrophy gene product, and is a component of gems

The survival of motor neurons (SMN) gene is the disease gene of spinal muscular atrophy (SMA), a common motor neuron degenerative disease. The SMN protein is part of a complex containing several proteins, of which one, SIP1 (SMN interacting protein 1), has been characterized so far. The SMN complex is found in both the cytoplasm and in the nucleus, where it is concentrated in bodies called gems. In the cytoplasm, SMN and SIP1 interact with the Sm core proteins of spliceosomal small nuclear ribonucleoproteins (snRNPs), and they play a critical role in snRNP assembly. In the nucleus, SMN is required for pre-mRNA splicing, likely by serving in the regeneration of snRNPs. Here, we report the identification of another component of the SMN complex, a novel DEAD box putative RNA helicase, named Gemin3. Gemin3 interacts directly with SMN, as well as with SmB, SmD2, and SmD3. Immunolocalization studies using mAbs to Gemin3 show that it colocalizes with SMN in gems. Gemin3 binds SMN via its unique COOH-terminal domain, and SMN mutations found in some SMA patients strongly reduce this interaction. The presence of a DEAD box motif in Gemin3 suggests that it may provide the catalytic activity that plays a critical role in the function of the SMN complex on RNPs.     

Unrip is a component of SMN complexes active in snRNP assembly

A macromolecular complex containing survival of motor neurons (SMN), the spinal muscular atrophy protein, and Gemin2-7 interacts with Sm proteins and snRNAs to carry out the assembly of these components into spliceosomal small nuclear ribonucleoproteins (snRNPs). Here we report the characterization of unr-interacting protein (unrip), a GH-WD protein of unknown function, as a component of the SMN complex that interacts directly with Gemin6 and Gemin7. Unrip also binds a subset of Sm proteins, and unrip-containing SMN complexes are necessary and sufficient to mediate the assembly of spliceosomal snRNPs. These results demonstrate that unrip functions in the pathway of snRNP biogenesis and is a marker of cellular SMN complexes active in snRNP assembly.     

A role for complexes of survival of motor neurons (SMN) protein with gemins and profilin in neurite-like cytoplasmic extensions of cultured nerve cells

Spinal muscular atrophy (SMA) is caused by reduced levels of SMN (survival of motor neurons protein) and consequent loss of motor neurons. SMN is involved in snRNP transport and nuclear RNA splicing, but axonal transport of SMN has also been shown to occur in motor neurons. SMN also binds to the small actin-binding protein, profilin. We now show that SMN and profilin II co-localise in the cytoplasm of differentiating rat PC12 cells and in neurite-like extensions, especially at their growth cones. Many components of known SMN complexes were also found in these extensions, including gemin2 (SIP-1), gemin6, gemin7 and unrip (unr-interacting protein). Coilin p80 and Sm core protein immunoreactivity, however, were seen only in the nucleus. SMN is known to associate with beta-actin mRNA and specific hnRNPs in axons and in neurite extensions of cultured nerve cells, and SMN also stimulates neurite outgrowth in cultures. Our results are therefore consistent with SMN complexes, rather than SMN alone, being involved in the transport of actin mRNPs along the axon as in the transport of snRNPs into the nucleus by similar SMN complexes. Antisense knockdown of profilin I and II isoforms inhibited neurite outgrowth of PC12 cells and caused accumulation of SMN and its associated proteins in cytoplasmic aggregates. BIAcore studies demonstrated a high affinity interaction of SMN with profilin IIa, the isoform present in developing neurons. Pathogenic missense mutations in SMN, or deletion of exons 5 and 7, prevented this interaction. The interaction is functional in that SMN can modulate actin polymerisation in vitro by reducing the inhibitory effect of profilin IIa. This suggests that reduced SMN in SMA might cause axonal pathfinding defects by disturbing the normal regulation of microfilament growth by profilins.     

High-resolution X-ray and NMR structures of the SMN Tudor domain: conformational variation in the binding site for symmetrically dimethylated arginine residues

The SMN protein, which is linked to spinal muscular atrophy (SMA), plays an important role in the assembly of the spliceosomal small nuclear ribonucleoprotein complexes. This function requires binding of SMN to the arginine-glycine (RG) rich C-terminal tails of the Sm proteins, which contain symmetrically dimethylated arginine residues (sDMA) in vivo. Using NMR titrations, we show that the SMN Tudor domain recognizes these sDMAs in the methylated RG repeats. Upon complex formation a cluster of conserved aromatic residues in the SMN Tudor domain interacts with the sDMA methyl groups. We present two high resolution structures of the uncomplexed SMN Tudor domain, a 1.8A crystal structure and an NMR structure that has been refined against a large number of backbone and side-chain residual dipolar couplings. The backbone conformation of both structures is very similar, however, differences are observed for the cluster of conserved aromatic side-chains in the sDMA binding pocket. In order to validate these variations we introduce a novel application of residual dipolar couplings for aromatic rings. We show that structural information can be derived from aromatic ring residual dipolar couplings, even in the presence of internal motions such as ring flipping. These residual dipolar couplings and ring current shifts independently confirm that the SMN Tudor domain adopts two different conformations in the sDMA binding pocket. The observed structural variations may play a role for the recognition of sDMAs.     

De novo translation initiation on membrane-bound ribosomes as a mechanism for localization of cytosolic protein mRNAs to the endoplasmic reticulum

The specialized protein synthesis functions of the cytosol and endoplasmic reticulum compartments are conferred by the signal recognition particle (SRP) pathway, which directs the cotranslational trafficking of signal sequence-encoding mRNAs from the cytosol to the endoplasmic reticulum (ER). Although subcellular mRNA distributions largely mirror the binary pattern predicted by the SRP pathway model, studies in mammalian cells, yeast, and Drosophila have also demonstrated that cytosolic protein-encoding mRNAs are broadly represented on ER-bound ribosomes. A mechanism for such noncanonical mRNA localization remains, however, to be identified. Here, we examine the hypothesis that de novo translation initiation on ER-bound ribosomes serves as a mechanism for localizing cytosolic protein-encoding mRNAs to the ER. As a test of this hypothesis, we performed single molecule RNA fluorescence in situ hybridization studies of subcellular mRNA distributions and report that a substantial fraction of mRNAs encoding the cytosolic protein GAPDH resides in close proximity to the ER. Consistent with these data, analyses of subcellular mRNA and ribosome distributions in multiple cell lines demonstrated that cytosolic protein mRNA-ribosome distributions were strongly correlated, whereas signal sequence-encoding mRNA-ribosome distributions were divergent. Ribosome footprinting studies of ER-bound polysomes revealed a substantial initiation codon read density enrichment for cytosolic protein-encoding mRNAs. We also demonstrate that eukaryotic initiation factor 2α is bound to the ER via a salt-sensitive, ribosome-independent mechanism. Combined, these data support ER-localized translation initiation as a mechanism for mRNA recruitment to the ER.     

Cytoplasmic localization and the choice of ligand determine aggregate formation by androgen receptor with amplified polyglutamine stretch

Polyglutamine tract expansion in androgen receptor is a recognized cause of spinal and bulbar muscular atrophy (SBMA), an X-linked motor neuronopathy. Similar mutations have been identified in proteins associated with other neurodegenerative diseases. Recent studies have shown that amplified polyglutamine repeat stretches form cellular aggregates that may be markers for these neurodegenerative diseases. Here we describe conditions that lead to aggregate formation by androgen receptor with polyglutamine stretch amplification. In transfection experiments, the mutant, compared with the wild-type receptor, was delayed in its cytoplasmic-nuclear translocation and formed large cytoplasmic aggregates in the presence of androgen. The cytoplasmic environment appears crucial for this aggregation, since retention of both the wild-type and mutant receptors in this cellular compartment by the deletion of their nuclear localization signals resulted in massive aggregation. Conversely, rapid nuclear transport of both receptors brought about by deletion of their ligand binding domains did not result in aggregate formation. However, androgen antagonists that altered the conformation of the ligand binding domain and promoted varying rates of cytoplasmic-nuclear translocation all inhibited aggregate formation. This demonstrates that in addition to the cytoplasmic localization, a distinct contribution of the ligand binding domain of the receptor is necessary for the aggregation. The finding that antiandrogens inhibit aggregate formation may provide the basis for in vivo determination of the role of these structures in SBMA.     

The spinal muscular atrophy protein SMN affects Drosophila germline nuclear organization through the U body-P body pathway

Survival motor neuron protein (SMN) is the determining factor for the human neurodegenerative disease spinal muscular atrophy (SMA). SMN is critical for small nuclear ribonucleoprotein (snRNP) assembly. Using Drosophila oogenesis as a model system, we show that mutations in smn cause abnormal nuclear organization in nurse cells and oocytes. Germline and mitotic clonal analysis reveals that both nurse cells and oocytes require SMN to maintain normal organization of nuclear compartments including chromosomes, nucleoli, Cajal bodies and histone locus bodies. We previously found that SMN-containing U bodies invariably associate with P bodies (Liu, J. L., and Gall, J. G. (2007). U bodies are cytoplasmic structures that contain uridine-rich small nuclear ribonucleoproteins and associate with P bodies. Proc. Natl. Acad. Sci. U. S. A. 104, 11655-11659.). Multiple lines of evidence implicate SMN in the regulation of germline nuclear organization through the connection of U bodies and P bodies. Firstly, smn germline clones phenocopy mutations for two P body components, Cup and Ovarian tumour (Otu). Secondly, P body mutations disrupt SMN distribution and the organization of U bodies. Finally, mutations in smn disrupt the function and organization of U bodies and P bodies. Taken together, our results suggest that SMN is required for the functional integrity of the U body-P body pathway, which in turn is important for maintaining proper nuclear architecture.     

Characterization of the adaptor protein ARH expression in the brain and ARH molecular interactions

Previously, pA134 was identified as one of the mRNAs present in the squid giant axon. Comparative sequence analyses revealed that the pA134 gene product manifested significant similarity to the mammalian lipoprotein receptor adaptor protein also known as ARH (autosomal recessive hypercholesterolemia). ARH mRNA and protein displayed very similar pattern of expression throughout the mouse brain. Significant levels of expression were observed in cells with a predominantly neuronal profile in the cerebellum, brainstem, olfactory bulb, hippocampus, and cortex. A yeast two hybrid screen for ARH protein interactions in mouse brain identified the following binders: amyloid precursor-like protein 1, low density lipoprotein receptor-related protein (LRP) 1, LRP8, and GABA receptor-associated protein-like 1. The interactions of ARH with LRP1 and GABA receptor-associated protein-like 1 were subsequently verified by co-immunoprecipitation of the protein complexes from transfected human embryonic kidney cells. The presence of ARH mRNA in axon of primary sympathetic neurons was established by RT-PCR analyses and confirmed by in situ hybridization. Taken together, our data suggest that ARH is a multifunctional protein whose spectrum of function in the brain goes beyond the traditionally known metabolism of lipoproteins, and that ARH may be locally synthesized in the axon.     

Identification of cis elements which direct the localization of maternal mRNAs to the posterior pole of ascidian embryos

During ascidian embryogenesis, some mRNAs show clear localization at the posterior-most region. These postplasmic mRNAs are divided into two groups (type I and type II) according to their pattern of localization. To elucidate how these localization patterns are achieved, we attempted to identify the localization elements of these mRNAs. When in vitro synthesized postplasmic mRNAs were introduced into eggs, these mRNAs showed posterior localization similar to the endogenous mRNAs. The posterior localization of these mRNAs was mediated by their 3' untranslated regions (3' UTRs), as is the case for several localized Drosophila and Xenopus mRNAs. We identified smaller fragments of the 3' UTRs of HrWnt-5 and HrPOPK-1 mRNAs (type I) and HrPet-3 mRNA (type II) that were sufficient to direct green fluorescent protein mRNA to the posterior pole. For the localization of HrWnt-5 mRNA, two UG dinucleotide repetitive elements were essential. Motifs similar to these small elements also exist within the HrPOPK-1 mRNA localization element and 3' UTR of HrZF-1 mRNA, suggesting the conservation of localization elements among type I mRNAs. In contrast, the smallest sequence that suffices for the posterior localization of HrPet-3 (a type II mRNA) has different features from those of type I mRNAs; indeed, it does not have an identifiable critical element. This difference may distinguish type II mRNAs from type I mRNAs. These findings, especially the identification of the small localization element of HrWnt-5 mRNA, provide new insights into the localization of mRNAs during ascidian embryogenesis.     

Developing prolamine protein bodies are associated with the cortical cytoskeleton in rice endosperm cells

 The mRNAs that encode the prolamine storage proteins in rice (Oryza sativa L.) endosperm cells are enriched on the surface of the prolamine protein bodies (PBs), a subcellular structure consisting of a prolamine intracisternal granule surrounded by rough endoplasmic reticulum membrane. Previous biochemical studies (D.G. Muench et al., 1998, Plant Physiol. 116: 559–569) have shown that prolamine mRNAs may be anchored to the PB surface via the cytoskeleton. To better understand the mechanism and role of mRNA localization in rice endosperm cells, we studied the subcellular development of prolamine PBs and their relationship with the cytoskeleton in rice endosperm cells. Confocal microscopy of endosperm cells showed that, unlike the glutelin PBs, the developing prolamine PBs are not randomly distributed within the cell, but instead are often enriched in the cortical region of the cell only a few micrometers beneath the plasma membrane. In addition, the peripheral prolamine PBs are closely associated with the cortical microtubule and actin filament networks. The cortical enrichment of rice prolamine protein bodies represents a unique example of endoplasmic reticulum subdomain localization in plant cells. The interaction of this endoplasmic reticulum subdomain with the cytoskeleton provides new insights on the possible mechanism and role of mRNA localization in plants. Received: 30 September 1999 / Accepted: 12 November 1999     

Caenorhabditis elegans in the study of SMN-interacting proteins: a role for SMI-1, an orthologue of human Gemin2 and the identification of novel components of the SMN complex

Spinal muscular atrophy is a common neuromuscular disorder caused by mutations in the survival motor neuron (SMN) gene. In mammals, SMN is tightly associated with Gemin2. To gain further insight into the functions of SMN and Gemin2, we have cloned and sequenced smi-1 (Survival of Motor neuron-Interacting protein 1), a C. elegans homologue of the human Gemin2 gene. We show that the SMI-1 expression pattern and RNA interference phenotype show considerable overlap with that previously reported for SMN-1. Finally, we demonstrate that the SMN-1 and SMI-1 proteins directly interact. Having demonstrated the utility of the C. elegans genetic model for investigating genes encoding SMN-interacting proteins, we have undertaken a yeast two-hybrid screen of a C. elegans cDNA library to identify novel proteins that interact with SMN-1. We show the direct interaction of SMN-1 with nine novel proteins, several of which may be involved in RNA metabolism.     

RNA immunoprecipitation and microarray analysis show a chloroplast Pentatricopeptide repeat protein to be associated with the 5' region of mRNAs whose translation it activates

Plant nuclear genomes encode hundreds of predicted organellar RNA binding proteins, few of which have been connected with their physiological RNA substrates and functions. In fact, among the largest family of putative RNA binding proteins in plants, the pentatricopeptide repeat (PPR) family, no physiologically relevant RNA ligands have been firmly established. We used the chloroplast-splicing factor CAF1 to demonstrate the fidelity of a microarray-based method for identifying RNAs associated with specific proteins in chloroplast extract. We then used the same method to identify RNAs associated with the maize (Zea mays) PPR protein CRP1. Two mRNAs whose translation is CRP1-dependent were strongly and specifically enriched in CRP1 coimmunoprecipitations. These interactions establish CRP1 as a translational regulator by showing that the translation defects in crp1 mutants are a direct consequence of the absence of CRP1. Additional experiments localized these interactions to the 5' untranslated regions and suggested a possible CRP1 interaction motif. These results enhance understanding of the PPR protein family by showing that a PPR protein influences gene expression through association with specific mRNAs in vivo, suggesting an unusual mode of RNA binding for PPR proteins, and highlighting the possibility that translational regulation may be a particularly common function of PPR proteins. Analogous methods should have broad application for the study of native RNA-protein interactions in both mitochondria and chloroplasts.     

Excitatory amino acid stimulation of the survival of rat cerebellar granule cells in culture is associated with an increase in SMN, the spinal muscular atrophy disease gene product

Summary. Excitatory amino acids which promote the survival of cerebellar granule cells in culture, also promote the expression of the survival of motor neuron (SMN) protein. Immunolocalization studies using SMN monoclonal antibody showed that SMN is decreased in cultures grown in low K⁺ or chemically defined medium with respect to cultures grown in high K⁺ medium and that an increase of SMN can be induced by treatment of low K⁺ cultures with glutamate or N-methyl-D-aspartate. Received March 31, 1999