运动神经元生存蛋白SMN与Sm蛋白的结合作用

脊髓性肌萎缩症（spinal muscular atrophy,SMA）是一类与运动神经元存活基因（survival of motor neurons gene,SMN gene）突变有关的神经系统变性疾病,而SMN基因的转录产物即为SMN蛋白（survival of motorneurons protein,SMN protein）。SMN蛋白与多种蛋白结合后发挥作用,如SMN-Sm蛋白的相互作用在富含尿嘧啶的小核核糖核蛋白体（uridine—richsmallribonucleo—proteins,UsnRNPs）转运装配中有重要意义。SMN蛋白是通过其Tudor结构域与剪接体sm蛋白的二甲基化修饰的富含精氨酸一氨基乙酸域（ar—ginineandglycine—rich,RG）结合。     

pICln Inhibits snRNP Biogenesis by Binding Core Spliceosomal Proteins

The U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins (snRNPs) form essential components of spliceosomes, the machinery that removes introns from pre-mRNAs in eukaryotic cells. A critical initial step in the complex process of snRNP biogenesis is the assembly of a group of common core proteins (Sm proteins) on spliceosomal snRNA. In this study we show by multiple independent methods that the protein pICln associates with Sm proteins in vivo and in vitro. The binding of pICln to Sm proteins interferes with Sm protein assembly on spliceosomal snRNAs and inhibits import of snRNAs into the nucleus. Furthermore, pICln prevents the interaction of Sm proteins with the survival of motor neurons (SMN) protein, an interaction that has been shown to be critical for snRNP biogenesis. These findings lead us to propose a model in which pICln participates in the regulation of snRNP biogenesis, at least in part by interfering with Sm protein interaction with SMN protein.     

snRNAs contain specific SMN-binding domains that are essential for snRNP assembly

To serve in its function as an assembly machine for spliceosomal small nuclear ribonucleoprotein particles (snRNPs), the survival of motor neurons (SMN) protein complex binds directly to the Sm proteins and the U snRNAs. A specific domain unique to U1 snRNA, stem-loop 1 (SL1), is required for SMN complex binding and U1 snRNP Sm core assembly. Here, we show that each of the major spliceosomal U snRNAs (U2, U4, and U5), as well as the minor splicing pathway U11 snRNA, contains a domain to which the SMN complex binds directly and with remarkable affinity (low nanomolar concentration). The SMN-binding domains of the U snRNAs do not have any significant nucleotide sequence similarity yet they compete for binding to the SMN complex in a manner that suggests the presence of at least two binding sites. Furthermore, the SMN complex-binding domain and the Sm site are both necessary and sufficient for Sm core assembly and their relative positions are critical for snRNP assembly. These findings indicate that the SMN complex stringently scrutinizes RNAs for specific structural features that are not obvious from the sequence of the RNAs but are required for their identification as bona fide snRNAs. It is likely that this surveillance capacity of the SMN complex ensures assembly of Sm cores on the correct RNAs only and prevents illicit, potentially deleterious, assembly of Sm cores on random RNAs.     

Sequence-specific interaction of U1 snRNA with the SMN complex

The survival of motor neurons (SMN) protein complex functions in the biogenesis of spliceosomal small nuclear ribonucleoprotein particles (snRNPs) and prob ably other RNPs. All spliceosomal snRNPs have a common core of seven Sm proteins. To mediate the assembly of snRNPs, the SMN complex must be able to bring together Sm proteins with U snRNAs. We showed previously that SMN and other components of the SMN complex interact directly with several Sm proteins. Here, we show that the SMN complex also interacts specifically with U1 snRNA. The stem--loop 1 domain of U1 (SL1) is necessary and sufficient for SMN complex binding in vivo and in vitro. Substitution of three nucleotides in the SL1 loop (SL1A3) abolishes SMN interaction, and the corresponding U1 snRNA (U1A3) is impaired in U1 snRNP biogenesis. Microinjection of excess SL1 but not SL1A3 into Xenopus oocytes inhibits SMN complex binding to U1 snRNA and U1 snRNP assembly. These findings indicate that SMN complex interaction with SL1 is sequence-specific and critical for U1 snRNP biogenesis, further supporting the direct role of the SMN complex in RNP biogenesis.     

Reconstitution of the human U snRNP assembly machinery reveals stepwise Sm protein organization

The assembly of spliceosomal U snRNPs depends on the coordinated action of PRMT5 and SMN complexes in vivo. These trans‐acting factors enable the faithful delivery of seven Sm proteins onto snRNA and the formation of the common core of snRNPs. To gain mechanistic insight into their mode of action, we reconstituted the assembly machinery from recombinant sources. We uncover a stepwise and ordered formation of distinct Sm protein complexes on the PRMT5 complex, which is facilitated by the assembly chaperone pICln. Upon completion, the formed pICln‐Sm units are displaced by new pICln‐Sm protein substrates and transferred onto the SMN complex. The latter acts as a Brownian machine that couples spontaneous conformational changes driven by thermal energy to prevent mis‐assembly and to ensure the transfer of Sm proteins to cognate RNA. Investigation of mutant SMN complexes provided insight into the contribution of individual proteins to these activities. The biochemical reconstitution presented here provides a basis for a detailed molecular dissection of the U snRNP assembly reaction.     

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.     

An assembly chaperone collaborates with the SMN complex to generate spliceosomal SnRNPs

Spliceosomal small nuclear ribonucleoproteins (snRNPs) are essential components of the nuclear pre-mRNA processing machinery. A hallmark of these particles is a ring-shaped core domain generated by the binding of Sm proteins onto snRNA. PRMT5 and SMN complexes mediate the formation of the core domain in vivo. Here, we have elucidated the mechanism of this reaction by both biochemical and structural studies. We show that pICln, a component of the PRMT5 complex, induces the formation of an otherwise unstable higher-order Sm protein unit. In this state, the Sm proteins are kinetically trapped, preventing their association with snRNA. The SMN complex subsequently binds to these Sm protein units, dissociates pICln, and catalyzes ring closure on snRNA. Our data identify pICln as an assembly chaperone and the SMN complex as a catalyst of spliceosomal snRNP formation. The mode of action of this combined chaperone/catalyst system is reminiscent of the mechanism employed by DNA clamp loaders.     

Methylation of Sm proteins by a complex containing PRMT5 and the putative U snRNP assembly factor pICln

Seven Sm proteins, termed B/B', D1, D2, D3, E, F, and G, assemble in an ordered manner onto U snRNAs to form the Sm core of the spliceosomal snRNPs U1, U2, U4/U6, and U5. The survival of motor neuron (SMN) protein binds to Sm proteins and mediates in the context of a macromolecular (SMN-) complex the assembly of the Sm core. Binding of SMN to Sm proteins is enhanced by modification of specific arginine residues in the Sm proteins D1 and D3 to symmetrical dimethylarginines (sDMAs), suggesting that assembly might be regulated at the posttranslational level. Here we provide evidence that the previously described pICln-complex, consisting of Sm proteins, the methyltransferase PRMT5, pICln, and two novel factors, catalyzes the sDMA modification of Sm proteins. In vitro studies further revealed that the pICln complex inhibits the spontaneous assembly of Sm proteins onto a U snRNA. This effect is mediated by pICln via its binding to the Sm fold of Sm proteins, thereby preventing specific interactions between Sm proteins required for the formation of the Sm core. Our data suggest that the pICln complex regulates an early step in the assembly of U snRNPs, possibly the transfer of Sm proteins to the SMN-complex.     

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.     

Toward an assembly line for U7 snRNPs: interactions of U7-specific Lsm proteins with PRMT5 and SMN complexes

The survival of motor neurons (SMN) complex mediates the assembly of small nuclear ribonucleoproteins (snRNPs) involved in splicing and histone RNA processing. A crucial step in this process is the binding of Sm proteins onto the SMN protein. For Sm B/B', D1, and D3, efficient binding to SMN depends on symmetrical dimethyl arginine (sDMA) modifications of their RG-rich tails. This methylation is achieved by another entity, the PRMT5 complex. Its pICln subunit binds Sm proteins whereas the PRMT5 subunit catalyzes the methylation reaction. Here, we provide evidence that Lsm10 and Lsm11, which replace the Sm proteins D1 and D2 in the histone RNA processing U7 snRNPs, associate with pICln in vitro and in vivo without receiving sDMA modifications. This implies that the PRMT5 complex is involved in an early stage of U7 snRNP assembly and hence may have a second snRNP assembly function unrelated to sDMA modification. We also show that the binding of Lsm10 and Lsm11 to SMN is independent of any methylation activity. Furthermore, we present evidence for two separate binding sites in SMN for Sm/Lsm proteins. One recognizes Sm domains and the second one, the sDMA-modified RG-tails, which are present only in a subset of these proteins.     

Gemin8 is required for the architecture and function of the survival motor neuron complex

The biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) in higher eukaryotes requires the functions of several cellular proteins and includes nuclear as well as cytoplasmic phases. In the cytoplasm, a macromolecular complex containing the survival motor neuron (SMN) protein, Gemin2-8 and Unrip mediates the ATP-dependent assembly of Sm proteins and snRNAs into snRNPs. To carry out snRNP assembly, the SMN complex binds directly to both Sm proteins and snRNAs; however, the contribution of the individual components of the SMN complex to its composition, interactions, and function is poorly characterized. Here, we have investigated the functional role of Gemin8 using novel monoclonal antibodies against components of the SMN complex and RNA interference experiments. We show that Gemin6, Gemin7, and Unrip form a stable cytoplasmic complex whose association with SMN requires Gemin8. Gemin8 binds directly to SMN and mediates its interaction with the Gemin6/Gemin7 heterodimer. Importantly, loss of Gemin6, Gemin7, and Unrip interaction with SMN as a result of Gemin8 knockdown affects snRNP assembly by impairing the SMN complex association with Sm proteins but not with snRNAs. These results reveal the essential role of Gemin8 for the proper structural organization of the SMN complex and the involvement of the heteromeric subunit containing Gemin6, Gemin7, Gemin8, and Unrip in the recruitment of Sm proteins to the snRNP assembly pathway.     

Specific sequence features, recognized by the SMN complex, identify snRNAs and determine their fate as snRNPs

The survival of motor neurons (SMN) complex is essential for the biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) as it binds to and delivers Sm proteins for assembly of Sm cores on the abundant small nuclear RNAs (snRNAs). Using the conserved snRNAs encoded by the lymphotropic Herpesvirus saimiri (HVS), we determined the specific sequence and structural features of RNAs for binding to the SMN complex and for Sm core assembly. We show that the minimal SMN complex-binding domain in snRNAs, except U1, is comprised of an Sm site (AUUUUUG) and an adjacent 3' stem-loop. The adenosine and the first and third uridines of the Sm site are particularly critical for binding of the SMN complex, which directly contacts the backbone phosphates of these uridines. The specific sequence of the adjacent stem (7 to 12 base pairs)-loop (4 to 17 nucleotides) is not important for SMN complex binding, but it must be located within a short distance of the 3' end of the RNA for an Sm core to assemble. Importantly, these defining characteristics are discerned by the SMN complex and not by the Sm proteins, which can bind to and assemble on an Sm site sequence alone. These findings demonstrate that the SMN complex is the identifier, as well as assembler, of the abundant class of snRNAs in cells because it is able to recognize an snRNP code that they contain.     

Coilin phosphorylation mediates interaction with SMN and SmB′

Cajal bodies (CBs) are subnuclear domains that participate in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and play a part in the assembly of the spliceosomal complex. The CB marker protein, coilin, interacts with survival of motor neuron (SMN) and Sm proteins. Several coilin phosphoresidues have been identified by mass spectrometric analysis. Phosphorylation of coilin affects its self-interaction and localization in the nucleus. We hypothesize that coilin phosphorylation also impacts its binding to SMN and Sm proteins. In vitro binding studies with a C-terminal fragment of coilin and corresponding phosphomimics show that SMN binds preferentially to dephosphorylated analogs and that SmB′ binds preferentially to phosphomimetic constructs. Bacterially expressed full-length coilin binds more SMN and SmB′ than does the C-terminal fragment. Co-immunoprecipitation and phosphatase experiments show that SMN also binds dephosphorylated coilin in vivo. These data show that phosphorylation of coilin influences interaction with its target proteins and, thus, may be significant in managing the flow of snRNPs through the CB.     

Lymphotropic Herpesvirus saimiri uses the SMN complex to assemble Sm cores on its small RNAs

The lymphotropic Herpesvirus saimiri (HVS) causes acute leukemia, T-cell lymphoma, and death in New World monkeys. HVS encodes seven small RNAs (HSURs) of unknown function. The HSURs acquire host Sm proteins and assemble Sm cores similar to those found on the spliceosomal small nuclear RNPs (snRNPs). Here we show that, like host snRNPs, HSURs use the SMN (survival of motor neurons) complex to assemble Sm cores. The HSURs bind the SMN complex directly and with very high affinity, similar to or higher than that of host snRNAs, and can outcompete host snRNAs for SMN-dependent assembly into RNPs. These observations highlight the general utility of the SMN complex for RNP assembly and suggest that infectious agents that engage the SMN complex may burden SMN-dependent pathways, possibly leading to a deleterious reduction in available SMN complex for essential host functions.     

SMN tudor domain structure and its interaction with the Sm proteins

Spinal muscular atrophy (SMA) is a common motor neuron disease that results from mutations in the Survival of Motor Neuron (SMN) gene. The SMN protein plays a crucial role in the assembly of spliceosomal uridine-rich small nuclear ribonucleoprotein (U snRNP) complexes via binding to the spliceosomal Sm core proteins. SMN contains a central Tudor domain that facilitates the SMN-Sm protein interaction. A SMA-causing point mutation (E134K) within the SMN Tudor domain prevents Sm binding. Here, we have determined the three-dimensional structure of the Tudor domain of human SMN. The structure exhibits a conserved negatively charged surface that is shown to interact with the C-terminal Arg and Gly-rich tails of Sm proteins. The E134K mutation does not disrupt the Tudor structure but affects the charge distribution within this binding site. An intriguing structural similarity between the Tudor domain and the Sm proteins suggests the presence of an additional binding interface that resembles that in hetero-oligomeric complexes of Sm proteins. Our data provide a structural basis for a molecular defect underlying SMA.     

Gemin8 is a novel component of the survival motor neuron complex and functions in small nuclear ribonucleoprotein assembly

The survival motor neuron (SMN) protein is the product of the spinal muscular atrophy disease gene. SMN and Gemin2-7 proteins form a large macromolecular complex that localizes in the cytoplasm as well as in the nucleoplasm and in nuclear Gems. The SMN complex interacts with several additional proteins and likely functions in multiple cellular pathways. In the cytoplasm, a subset of SMN complexes containing unrip and Sm proteins mediates the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). Here, by mass spectrometry analysis of SMN complexes purified from HeLa cells, we identified a novel protein that is evolutionarily conserved in metazoans, and we named it Gemin8. Co-immunoprecipitation and immunolocalization experiments demonstrated that Gemin8 is associated with the SMN complex and is localized in the cytoplasm and in the nucleus, where it is highly concentrated in Gems. Gemin8 interacts directly with the Gemin6-Gemin7 heterodimer and, together with unrip, these proteins form a heteromeric subunit of the SMN complex. Gemin8 is also associated with Sm proteins, and Gemin8-containing SMN complexes are competent to carry out snRNP assembly. Importantly, RNA interference experiments indicate that Gemin8 knock-down impairs snRNP assembly, and Gemin8 expression is down-regulated in cells with low levels of SMN. These results demonstrate that Gemin8 is a novel integral component of the SMN complex and extend the repertoire of cellular proteins involved in the pathway of snRNP biogenesis.     

UsnRNP biogenesis: mechanisms and regulation

Macromolecular complexes composed of proteins or proteins and nucleic acids rather than individual macromolecules mediate many cellular activities. Maintenance of these activities is essential for cell viability and requires the coordinated production of the individual complex components as well as their faithful incorporation into functional entities. Failure of complex assembly may have fatal consequences and can cause severe diseases. While many macromolecular complexes can form spontaneously in vitro, they often require aid from assembly factors including assembly chaperones in the crowded cellular environment. The assembly of RNA protein complexes implicated in the maturation of pre-mRNAs (termed UsnRNPs) has proven to be a paradigm to understand the action of assembly factors and chaperones. UsnRNPs are assembled by factors united in protein arginine methyltransferase 5 (PRMT5)- and survival motor neuron (SMN)-complexes, which act sequentially in the UsnRNP production line. While the PRMT5-complex pre-arranges specific sets of proteins into stable intermediates, the SMN complex displaces assembly factors from these intermediates and unites them with UsnRNA to form the assembled RNP. Despite advanced mechanistic understanding of UsnRNP assembly, our knowledge of regulatory features of this essential and ubiquitous cellular function remains remarkably incomplete. One may argue that the process operates as a default biosynthesis pathway and does not require sophisticated regulatory cues. Simple theoretical considerations and a number of experimental data, however, indicate that regulation of UsnRNP assembly most likely happens at multiple levels. This review will not only summarize how individual components of this assembly line act mechanistically but also why, how, and when the UsnRNP workflow might be regulated by means of posttranslational modification in response to cellular signaling cues.     

The SMN complex is associated with snRNPs throughout their cytoplasmic assembly pathway

The common neurodegenerative disease spinal muscular atrophy is caused by reduced levels of the survival of motor neurons (SMN) protein. SMN associates with several proteins (Gemin2 to Gemin6) to form a large complex which is found both in the cytoplasm and in the nucleus. The SMN complex functions in the assembly and metabolism of several RNPs, including spliceosomal snRNPs. The snRNP core assembly takes place in the cytoplasm from Sm proteins and newly exported snRNAs. Here, we identify three distinct cytoplasmic SMN complexes, each representing a defined intermediate in the snRNP biogenesis pathway. We show that the SMN complex associates with newly exported snRNAs containing the nonphosphorylated form of the snRNA export factor PHAX. The second SMN complex identified contains assembled Sm cores and m(3)G-capped snRNAs. Finally, the SMN complex is associated with a preimport complex containing m(3)G-capped snRNP cores bound to the snRNP nuclear import mediator snurportin1. Thus, the SMN complex is associated with snRNPs during the entire process of their biogenesis in the cytoplasm and may have multiple functions throughout this process.     

Why do cells need an assembly machine for RNA-protein complexes?

Small nuclear ribonucleoproteins (snRNPs) are crucial for pre-mRNA processing to mRNAs. Each snRNP contains a small nuclear RNA (snRNA) and an extremely stable core of seven Sm proteins. The snRNP biogenesis pathway is complex, involving nuclear export of snRNA, Sm-core assembly in the cytoplasm and re-import of the mature snRNP. Although in vitro Sm cores assemble readily on uridine-rich RNAs, the assembly in cells is carried out by the survival of motor neurons (SMN) complex. The SMN complex stringently scrutinizes RNAs for specific features that define them as snRNAs and identifies the RNA-binding Sm proteins. We discuss how this surveillance capacity of the SMN complex might ensure assembly of Sm cores only on the correct RNAs and prevent illicit, potentially deleterious assembly of Sm cores on random RNAs.     

Purification of native survival of motor neurons complexes and identification of Gemin6 as a novel component.

The survival of motor neurons (SMN) protein, the product of the gene responsible for the motor neuron degenerative disease spinal muscular atrophy (SMA), is part of a large macromolecular complex. The SMN complex is localized in both the cytoplasm and the nucleus and contains SMN, Gemin2, Gemin3, Gemin4, Gemin5, and a few not yet identified proteins. The SMN complex plays a key role in the biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and other ribonucleoprotein particles. As a step toward the complete characterization of the components of the SMN complex, we generated stable cell lines that express FLAG-tagged SMN or Gemin2 under the control of a tetracycline-inducible promoter. Native SMN complexes of identical protein composition to those isolated by immunoprecipitation with anti-SMN antibodies were purified by affinity chromatography from extracts of both cell lines. Here we report the identification by mass spectrometry of a novel protein component of the SMN complex termed Gemin6. Co-immunoprecipitation, immunolocalization, and in vitro binding experiments demonstrate that Gemin6 is a component of the SMN complex that localizes to gems and interacts with several Sm proteins of the spliceosomal snRNPs.