Analysis of SMN-neurite granules: Core Cajal body components are absent from SMN-cytoplasmic complexes

Childhood spinal muscular atrophy (SMA) is caused by a reduction in survival motor neuron (SMN) protein. SMN is a ubiquitously expressed house keeping protein that is involved in RNA production and processing. However, although SMN is expressed in every cell type, only the lower motor neurons of the spinal cord are degraded in SMA. It remains unclear why this is the case. Recently, SMN has been linked to the axonal transport of β-actin mRNA from the cell body down to the growth cones. β-Actin is transported actively in neurite granules (NGs). However, it remains unclear which known SMN-binding partners are present in these SMN-NGs. To address this we have analysed SMN-NGs in a human neuronal cell line, SH-SY5Y, using antibodies against the majority of reported SMN-binding partners, including: Gemin2, Gemin3, Gemin4, Gemin5, Gemin6, Gemin7, Sm core proteins, fibrillarin, EWS, PFNII, Unrip and ZPR1. The obtained results highlight the metamorphic nature of the SMN complex, suggesting that not all the “core” SMN-binding proteins are transported in SMN-NGs.     

Detection of snRNP assembly intermediates in Cajal bodies by fluorescence resonance energy transfer

Spliceosomal small nuclear ribonucleoprotein particles (snRNPs) are required for pre-mRNA splicing throughout the nucleoplasm, yet snRNPs also concentrate in Cajal bodies (CBs). To address a proposed role of CBs in snRNP assembly, we have used fluorescence resonance energy transfer (FRET) microscopy to investigate the subnuclear distribution of specific snRNP intermediates. Two distinct complexes containing the protein SART3 (p110), required for U4/U6 snRNP assembly, were localized: SART3.U6 snRNP and SART3.U4/U6 snRNP. These complexes segregated to different nuclear compartments, with SART3.U6 snRNPs exclusively in the nucleoplasm and SART3.U4/U6 snRNPs preferentially in CBs. Mutant cells lacking the CB-specific protein coilin and consequently lacking CBs exhibited increased nucleoplasmic levels of SART3.U4/U6 snRNP complexes. Reconstitution of CBs in these cells by expression of exogenous coilin restored accumulation of SART3.U4/U6 snRNP in CBs. Thus, while some U4/U6 snRNP assembly can occur in the nucleoplasm, these data provide evidence that SART3.U6 snRNPs form in the nucleoplasm and translocate to CBs where U4/U6 snRNP assembly occurs.     

Cajal bodies and coilin--moving towards function

Many nuclear factors are concentrated within nonmembrane-bound subnuclear bodies. The Cajal body is an example of a conserved nuclear compartment that has been linked to molecular disease. Recent studies have shown Cajal bodies to be surprisingly mobile and offer clues about their function in the cell.     

The spinal muscular atrophy disease gene product, SMN: A link between snRNP biogenesis and the Cajal (coiled) body

The spliceosomal snRNAs U1, U2, U4, and U5 are synthesized in the nucleus, exported to the cytoplasm to assemble with Sm proteins, and reimported to the nucleus as ribonucleoprotein particles. Recently, two novel proteins involved in biogenesis of small nuclear ribonucleoproteins (snRNPs) were identified, the Spinal muscular atrophy disease gene product (SMN) and its associated protein SIP1. It was previously reported that in HeLa cells, SMN and SIP1 form discrete foci located next to Cajal (coiled) bodies, the so-called "gemini of coiled bodies" or "gems." An intriguing feature of gems is that they do not appear to contain snRNPs. Here we show that gems are present in a variable but small proportion of rapidly proliferating cells in culture. In the vast majority of cultured cells and in all primary neurons analyzed, SMN and SIP1 colocalize precisely with snRNPs in the Cajal body. The presence of SMN and SIP1 in Cajal bodies is confirmed by immunoelectron microscopy and by microinjection of antibodies that interfere with the integrity of the structure. The association of SMN with snRNPs and coilin persists during cell division, but at the end of mitosis there is a lag period between assembly of new Cajal bodies in the nucleus and detection of SMN in these structures, suggesting that SMN is targeted to preformed Cajal bodies. Finally, treatment of cells with leptomycin B (a drug that blocks export of U snRNAs to the cytoplasm and consequently import of new snRNPs into the nucleus) is shown to deplete snRNPs (but not SMN or SIP1) from the Cajal body. This suggests that snRNPs flow through the Cajal body during their biogenesis pathway.     

Symmetrical dimethylarginine methylation is required for the localization of SMN in Cajal bodies and pre-mRNA splicing

The nuclear structures that contain symmetrical dimethylated arginine (sDMA)-modified proteins and the role of this posttranslational modification is unknown. Here we report that the Cajal body is a major epitope in HeLa cells for an sDMA-specific antibody and that coilin is an sDMA-containing protein as analyzed by using the sDMA-specific antibody and matrix-assisted laser desorption ionization time of flight mass spectrometry. The methylation inhibitor 5'-deoxy-5'-methylthioadenosine reduces the levels of coilin methylation and causes the appearance of SMN-positive gems. In cells devoid of Cajal bodies, such as primary fibroblasts, sDMA-containing proteins concentrated in speckles. Cells from a patient with spinal muscular atrophy, containing low levels of the methyl-binding protein SMN, localized sDMA-containing proteins in the nucleoplasm as a discrete granular pattern. Splicing reactions are efficiently inhibited by using the sDMA-specific antibody or by using hypomethylated nuclear extracts, showing that active spliceosomes contain sDMA polypeptides and suggesting that arginine methylation is important for efficient pre-mRNA splicing. Our findings support a model in which arginine methylation is important for the localization of coilin and SMN in Cajal bodies.     

SUMO-1 transiently localizes to Cajal bodies in mammalian neurons

Cajal bodies (CBs) are nuclear organelles involved in the maturation of small nuclear ribonucleoproteins required for the processing of pre-mRNAs. They concentrate coilin, splicing factors and the survival of motor neuron protein (SMN). By using immunocytochemistry and transfection experiments with GFP–SUMO-1, DsRed1-Ubc9, GFP–coilin and GFP–SMN constructs we demonstrate the presence of SUMO-1 and the SUMO conjugating enzyme (Ubc9) in a subset of CBs in undifferentiated neuron-like UR61 cells. Furthermore, SUMO-1 is transiently localized into neuronal CBs from adult nervous tissue in response to osmotic stress or inhibition of methyltransferase activity. SUMO-1-positive CBs contain coilin, SMN and small nuclear ribonucleoproteins, suggesting that they are functional CBs involved in pre-mRNA processing. Since coilin and SMN have several putative motifs of SUMO-1 modification, we suggest that the sumoylation of coilin and/or SMN might play a role in the molecular reorganization of CBs during the neuronal differentiation or stress–response.     

Localization of interchromatin granule cluster and Cajal body components in oocyte nuclear bodies of the hemipterans

An oocyte nucleus contains different extrachromosomal nuclear domains collectively called nuclear bodies (NBs). In the present work we revealed, using immunogold labeling electron microscopy, some marker components of interchromatin granule clusters (IGCs) and Cajal bodies (CBs) in morphologically heterogeneous oocyte NBs studied in three hemipteran species: Notostira elongata, Capsodes gothicus (Miridae) and Velia caprai (Veliidae). Both IGC and CB counterparts were revealed in oocyte nuclei of the studied species but morphological and biochemical criteria were found to be not sufficient to determine carefully the define type of oocyte NBs. We found that the molecular markers of the CBs (coilin and non-phosphorylated RNA polymerase II) and IGCs (SC35 protein) may be localized in the same NB. Anti-SC35 antibody may decorate not only a granular material representing true interchromatin granules but also masks some fibrillar parts of complex NBs. Our first observations on the hemipteran oocyte NBs confirm the high complexity and heterogeneity of insect oocyte IGCs and CBs in comparison with those in mammalian somatic cells and amphibian oocytes.     

The relationship between SMN, the spinal muscular atrophy protein, and nuclear coiled bodies in differentiated tissues and cultured cells

The spinal muscular atrophy protein, SMN, is a cytoplasmic protein that is also found in distinct nuclear structures called "gems." Gems are closely associated with nuclear coiled bodies and both may have a direct role in snRNP maturation and pre-RNA splicing. There has been some controversy over whether gems and coiled bodies colocalize or form adjacent/independent structures in HeLa and other cultured cells. Using a new panel of antibodies against SMN and antibodies against coilin-p80, a systematic and quantitative study of adult differentiated tissues has shown that gems always colocalize with coiled bodies. In some tissues, a small proportion of coiled bodies (<10%) had no SMN, but independent or adjacent gems were not found. The most striking observation, however, was that many cell types appear to have neither gems nor coiled bodies (e.g., cardiac and smooth muscle, blood vessels, stomach, and spleen) and this expression pattern is conserved across human, rabbit, and pig species. This shows that assembly of distinct nuclear bodies is not essential for RNA splicing and supports the view that they may be storage sites for reserves of essential proteins and snRNPs. Overexpression of SMN in COS-7 cells produced supernumerary nuclear bodies, most of which also contained coilin-p80, confirming the close relationship between gems and coiled bodies. However, when SMN is reduced to very low levels in type I SMA fibroblasts, coiled bodies are still formed. Overall, the data suggest that gem/coiled body formation is not determined by high cytoplasmic SMN concentrations or high metabolic activity alone and that a differentiation-specific factor may control their formation.     

Nuclear localization properties of a conserved protuberance in the Sm core complex

The nuclear import signal of snRNPs is composed of two essential components, the m(3)G cap structure of the snRNA and the Sm core NLS carried by the Sm protein core complex. We have previously proposed that, in yeast, this last determinant is represented by a basic-rich protuberance formed by the C-terminal extensions of Sm proteins. In mammals, as well as in other organisms, this component has not yet been precisely defined. Using GFP-Sm fusion constructs and immunolocalization as well as biochemical experiments, we show here that the C-terminal domains of human SmD1 and SmD3 proteins possess nuclear localization properties. Deletions of these domains increase cytoplasmic fluorescence and cytoplasmic localization of GFP-Sm mutant fusion alleles. Our results are consistent with a model in which the Sm core NLS is evolutionarily conserved and composed of a basic-rich protuberance formed by C-terminal domains of different Sm subtypes.     

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.     

In vivo analysis of Cajal body movement, separation, and joining in live human cells

Cajal bodies (also known as coiled bodies) are subnuclear organelles that contain specific nuclear antigens, including splicing small nuclear ribonucleoproteins (snRNPs) and a subset of nucleolar proteins. Cajal bodies are localized in the nucleoplasm and are often found at the nucleolar periphery. We have constructed a stable HeLa cell line, HeLa(GFP-coilin), that expresses the Cajal body marker protein, p80 coilin, fused to the green fluorescent protein (GFP-coilin). The localization pattern and biochemical properties of the GFP-coilin fusion protein are identical to the endogenous p80 coilin. Time-lapse recordings on 63 nuclei of HeLa(GFP-coilin) cells showed that all Cajal bodies move within the nucleoplasm. Movements included translocations through the nucleoplasm, joining of bodies to form larger structures, and separation of smaller bodies from larger Cajal bodies. Also, we observed Cajal bodies moving to and from nucleoli. The data suggest that there may be at least two classes of Cajal bodies that differ in their size, antigen composition, and dynamic behavior. The smaller size class shows more frequent and faster rates of movement, up to 0.9 microm/min. The GFP-coilin protein is dynamically associated with Cajal bodies as shown by changes in their fluorescence intensity over time. This study reveals an unexpectedly high level of movement and interactions of nuclear bodies in human cells and suggests that these movements may be driven, at least in part, by regulated mechanisms.     

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.     

内脏平滑肌Cajal间质细胞起搏功能(英文)

胃肠道的大部分区域都存在着一种特殊的间质细胞——Cajal间质细胞(interstitial cells of Cajal,ICCs)。尽管在100多年前它们的存在就已被发现,但是直到最近几十年的研究才逐渐揭示了它们的功能。在胃肠道,ICCs被认为是平滑肌自发性节律性电活动,即"基本电节律"(又称"慢波")的起搏细胞,并介导神经至平滑肌的神经信号传递活动。除胃肠道外,ICC样细胞同样存在于其它内脏平滑肌,如泌尿、生殖系统以及血管平滑肌等。本文仅就这些内脏平滑肌ICCs的功能做一简单综述。