Survival Motor Neuron Complex Research Articles

Gemin5, a Novel WD Repeat Protein Component of the SMN Complex That Binds Sm Proteins

The survival of motor neurons (SMN) protein is the product of the disease gene of spinal muscular atrophy and is found both in the cytoplasm and the nucleus, where it is concentrated in gems. SMN is part of a multi-protein complex that includes Gemin2, Gemin3, and Gemin4. The SMN complex plays an important role in the cytoplasmic assembly of small nuclear ribonucleoproteins (snRNPs) and likely other RNPs in pre-mRNA splicing and in the assembly of transcriptosomes. Here, we report the identification of an additional component of the SMN complex, a novel WD repeat protein termed Gemin5. Gemin5 binds SMN directly and is a component of the SMN complex. Furthermore, Gemin5 interacts with several of the snRNP core proteins including SmB, SmD1, SmD2, SmD3, and SmE, suggesting that it participates in the activities of the SMN complex in snRNP assembly. Immunolocalization studies demonstrate that Gemin5 is found in the cytoplasm and in the nucleus, where it colocalizes with SMN in gems. The presence of 13 WD repeat domains in the amino-terminal half of Gemin5 and a coiled-coil motif near its carboxyl terminus indicate that it may form a large heteromeric complex and engage in multiple interactions.

The methylosome, a 20S complex containing JBP1 and pICln, produces dimethylarginine-modified Sm proteins.

snRNPs, integral components of the pre-mRNA splicing machinery, consist of seven Sm proteins which assemble in the cytoplasm as a ring structure on the snRNAs U1, U2, U4, and U5. The survival motor neuron (SMN) protein, the spinal muscular atrophy disease gene product, is crucial for snRNP core particle assembly in vivo. SMN binds preferentially and directly to the symmetrical dimethylarginine (sDMA)-modified arginine- and glycine-rich (RG-rich) domains of SmD1 and SmD3. We found that the unmodified, but not the sDMA-modified, RG domains of SmD1 and SmD3 associate with a 20S methyltransferase complex, termed the methylosome, that contains the methyltransferase JBP1 and a JBP1-interacting protein, pICln. JBP1 binds SmD1 and SmD3 via their RG domains, while pICln binds the Sm domains. JBP1 produces sDMAs in the RG domain-containing Sm proteins. We further demonstrate the existence of a 6S complex that contains pICln, SmD1, and SmD3 but not JBP1. SmD3 from the methylosome, but not that from the 6S complex, can be transferred to the SMN complex in vitro. Together with previous results, these data indicate that methylation of Sm proteins by the methylosome directs Sm proteins to the SMN complex for assembly into snRNP core particles and suggest that the methylosome can regulate snRNP assembly.

SMN interacts with a novel family of hnRNP and spliceosomal proteins.

Spinal muscular atrophy (SMA) is a common neurodegenerative disease caused by deletion or loss-of-function mutations of the survival of motor neurons (SMN) protein. SMN is in a complex with several proteins, including Gemin2, Gemin3 and Gemin4, and it plays important roles in small nuclear ribonucleoprotein (snRNP) biogenesis and in pre-mRNA splicing. Here, we characterize three new hnRNP proteins, collectively referred to as hnRNP Qs, which are derived from alternative splicing of a single gene. The hnRNP Q proteins interact with SMN, and the most common SMN mutant found in SMA patients is defective in its interactions with them. We further demonstrate that hnRNP Qs are required for efficient pre-mRNA splicing in vitro. The hnRNP Q proteins may provide a molecular link between the SMN complex and splicing.

Residual Cajal bodies in coilin knockout mice fail to recruit Sm snRNPs and SMN, the spinal muscular atrophy gene product.

Cajal bodies (CBs) are nuclear suborganelles involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). In addition to snRNPs, they are highly enriched in basal transcription and cell cycle factors, the nucleolar proteins fibrillarin (Fb) and Nopp140 (Nopp), the survival motor neuron (SMN) protein complex, and the CB marker protein, p80 coilin. We report the generation of knockout mice lacking the COOH-terminal 487 amino acids of coilin. Northern and Western blot analyses demonstrate that we have successfully removed the full-length coilin protein from the knockout animals. Some homozygous mutant animals are viable, but their numbers are reduced significantly when crossed to inbred backgrounds. Analysis of tissues and cell lines from mutant animals reveals the presence of extranucleolar foci that contain Fb and Nopp but not other typical nucleolar markers. These so-called “residual” CBs neither condense Sm proteins nor recruit members of the SMN protein complex. Transient expression of wild-type mouse coilin in knockout cells results in formation of CBs and restores these missing epitopes. Our data demonstrate that full-length coilin is essential for proper formation and/or maintenance of CBs and that recruitment of snRNP and SMN complex proteins to these nuclear subdomains requires sequences within the coilin COOH terminus.

The survival of motor neurons (SMN) protein interacts with the snoRNP proteins fibrillarin and GAR1

Background: The survival of motor neurons (SMN) protein is the protein product of the spinal muscular atrophy (SMA) disease gene. SMN and its associated proteins Gemin2, Gemin3, and Gemin4 form a large complex that plays a role in snRNP assembly, pre-mRNA splicing, and transcription. The functions of SMN in these processes are mediated by a direct interaction of SMN with components of these machineries, such as Sm proteins and RNA helicase A.Results: We show that SMN binds directly to fibrillarin and GAR1. Fibrillarin and GAR1 are specific markers of the two classes of small nucleolar ribonucleoprotein particles (snoRNPs) that are involved in posttranscriptional processing and modification of ribosomal RNA. SMN interaction requires the arginine- and glycine-rich domains of both fibrillarin and GAR1 and is defective in SMN mutants found in some SMA patients. Coimmunoprecipitations demonstrate that the SMN complex associates with fibrillarin and with GAR1 in vivo. The inhibition of RNA polymerase I transcription causes a transient redistribution of SMN to the nucleolar periphery and loss of fibrillarin and GAR1 colocalization with SMN in gems. Furthermore, the expression of a dominant-negative mutant of SMN (SMNΔN27) causes snoRNPs to accumulate outside of the nucleolus in structures that also contain components of gems and coiled (Cajal) bodies.Conclusions: These findings identify fibrillarin and GAR1 as novel interactors of SMN and suggest a function for the SMN complex in the assembly and metabolism of snoRNPs. We propose that the SMN complex performs functions necessary for the biogenesis and function of diverse ribonucleoprotein complexes.

A Functional Interaction between the Survival Motor Neuron Complex and RNA Polymerase II

The survival motor neuron (SMN) protein, the protein product of the spinal muscular atrophy (SMA) disease gene, plays a role in the assembly and regeneration of small nuclear ribonucleoproteins (snRNPs) and spliceosomes. By nanoelectrospray mass spectrometry, we identified RNA helicase A (RHA) as an SMN complex-associated protein. RHA is a DEAH box RNA helicase which binds RNA polymerase II (pol II) and reportedly functions in transcription. SMN interacts with RHA in vitro, and this interaction is impaired in mutant SMNs found in SMA patients. Coimmunoprecipitation demonstrated that the SMN complex is associated with pol II, snRNPs, and RHA in vivo. In vitro experiments suggest that RHA mediates the association of SMN with the COOH-terminal domain of pol II. Moreover, transfection of cells with a dominant negative mutant of SMN, SMNDeltaN27, causes accumulation of pol II, snRNPs, and RHA in nuclear structures that contain the known markers of gems and coiled bodies, and inhibits RNA pol I and pol II transcription in vivo. These findings indicate a functional as well as physical association of the SMN complex with pol II and suggest a role for the SMN complex in the assembly of the pol II transcription/processing machinery.

Characterization of a nuclear 20S complex containing the survival of motor neurons (SMN) protein and a specific subset of spliceosomal Sm proteins.

Spinal muscular atrophy (SMA) is a neurodegenerative disease of motor neurons caused by reduced levels of functional survival of motor neurons (SMN) protein. Cytoplasmic SMN directly interacts with spliceosomal Sm proteins and facilitates their assembly onto U snRNAs. Nuclear SMN, in contrast, mediates recycling of pre-mRNA splicing factors. In this study, we have addressed the function of SMN in the nucleus. We show that a monoclonal antibody directed against SMN inhibits pre-mRNA splicing. Interestingly, the mode of inhibition suggests a novel role for SMN in splicing that occurs prior to, or in addition to, its role in recycling. Using biochemical fractionation and anti-SMN immunoaffinity chromatography, we identified two distinct nuclear SMN complexes termed NSC1 and NSC2. The biochemical properties and protein composition of NSC1 were determined in detail. NSC1 migrates in sucrose gradients as a U snRNA-free 20S complex containing at least 10 proteins. In addition to SMN, these include the SMN-interacting protein 1 (SIP-1), the putative helicase dp103/Gemin3, the novel dp103/Gemin3-interacting protein GIP1/Gemin4 and three additional proteins with apparent masses of 43, 33 and 18 kDa, respectively. Most surprisingly, NSC1 also contains a specific subset of spliceosomal Sm proteins. This shows that the SMN-Sm protein interaction is not restricted to the cytoplasm. Our data imply that nuclear SMN affects splicing by modulating the Sm protein composition of U snRNPs.

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.

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.