Coilin levels and modifications influence artificial reporter splicing

Cajal bodies (CBs) and Gems are nuclear domains that contain factors responsible for spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis. The marker protein for CBs is coilin. In addition to snRNPs, coilin and other factors, canonical CBs contain the survivor of motor neuron protein (SMN). SMN can also localize to Gems. Considering the important role that coilin plays in the formation and composition of CBs, we tested the splicing efficiency of several cell lines that vary in regards to coilin level and modification using an artificial reporter substrate. We found that cells with both hypomethylated coilin and Gems are more efficient at reporter splicing compared to cells in which SMN localizes to CBs. In contrast, coilin reduction, which induces Gem formation, decreases cell proliferation and artificial reporter splicing. These findings demonstrate that coilin modifications or levels impact artificial reporter splicing, possibly by influencing snRNP biogenesis. Received 26 December 2007; received after revision 5 February 2008; accepted 7 February 2008     

Immobile survival of motoneuron (SMN) protein stored in Cajal bodies can be mobilized by protein interactions

Reduced levels of survival of motoneuron (SMN) protein lead to spinal muscular atrophy, but it is still unknown how SMN protects motoneurons in the spinal cord against degeneration. In the nucleus, SMN is associated with two types of nuclear bodies denoted as gems and Cajal bodies (CBs). The 23 kDa isoform of fibroblast growth factor-2 (FGF-2²³) is a nuclear protein that binds to SMN and destabilizes the SMN-Gemin2 complex. In the present study, we show that FGF-2²³ depletes SMN from CBs without affecting their general structure. FRAP analysis of SMN-EGFP in CBs demonstrated that the majority of SMN in CBs remained mobile and allowed quantification of fast, slow and immobile nuclear SMN populations. The potential for SMN release was confirmed by in vivo photoconversion of SMN-Dendra2, indicating that CBs concentrate immobile SMN that could have a specialized function in CBs. FGF-2²³ accelerated SMN release from CBs, accompanied by a conversion of immobile SMN into a mobile population. Furthermore, FGF-2²³ caused snRNP accumulation in CBs. We propose a model in which Cajal bodies store immobile SMN that can be mobilized by its nuclear interaction partner FGF-2²³, leading to U4 snRNP accumulation in CBs, indicating a role for immobile SMN in tri-snRNP assembly.     

Modulation of SMN nuclear foci and cytoplasmic localization by its C-terminus

The survival of motor neuron ( SMN1) gene product, SMN, is detected both in the cytoplasm and in nuclear gems and cajal bodies. We show here that SMN exon 6 is essential both for formation of its nuclear foci and for its cytoplasmic localization. However, exon 7 inhibits the formation of SMN nuclear foci but promotes SMN cytoplasmic localization. More interestingly, we find that a random C-terminal tag of five or more amino acids downstream of exon 6 is sufficient to inhibit the occurrence of multiple nuclear foci and to promote cytoplasmic localization of SMNDelta7, the primary product of the SMN2 gene. Moreover, SMNDelta7 proteins that bear spinal muscular atrophy mutations in exon 6 either showed defects in nuclear foci formation or enhanced cytoplasmic localization. We conclude that exon 6 and exon 7 synergistically regulate SMN distribution that may require specific exon 6 motifs but is independent of specific sequences in exon 7.     

The role of survival motor neuron protein (SMN) in protein homeostasis

Ever since loss of survival motor neuron (SMN) protein was identified as the direct cause of the childhood inherited neurodegenerative disorder spinal muscular atrophy, significant efforts have been made to reveal the molecular functions of this ubiquitously expressed protein. Resulting research demonstrated that SMN plays important roles in multiple fundamental cellular homeostatic pathways, including a well-characterised role in the assembly of the spliceosome and biogenesis of ribonucleoproteins. More recent studies have shown that SMN is also involved in other housekeeping processes, including mRNA trafficking and local translation, cytoskeletal dynamics, endocytosis and autophagy. Moreover, SMN has been shown to influence mitochondria and bioenergetic pathways as well as regulate function of the ubiquitin–proteasome system. In this review, we summarise these diverse functions of SMN, confirming its key role in maintenance of the homeostatic environment of the cell.     

The special Sm core structure of the U7 snRNP: far-reaching significance of a small nuclear ribonucleoprotein

The polypeptide composition of the U7 small nuclear ribonucleoprotein (snRNP) involved in histone messenger RNA (mRNA) 3' end formation has recently been elucidated. In contrast to spliceosomal snRNPs, which contain a ring-shaped assembly of seven so-called Sm proteins, in the U7 snRNP the Sm proteins D1 and D2 are replaced by U7-specific Sm-like proteins, Lsm10 and Lsm11. This polypeptide composition and the unusual structure of Lsm11, which plays a role in histone RNA processing, represent new themes in the biology of Sm/Lsm proteins. Moreover this structure has important consequences for snRNP assembly that is mediated by two complexes containing the PRMT5 methyltransferase and the SMN (survival of motor neurons) protein, respectively. Finally, the ability to alter this polypeptide composition by a small mutation in U7 snRNA forms the basis for using modified U7 snRNA derivatives to alter specific pre-mRNA splicing events, thereby opening up a new way for antisense gene therapy.     

Friedrich Miescher Prize awardee lecture review. A conserved family of nuclear export receptors mediates the exit of messenger RNA to the cytoplasm

The distinguishing feature of eukaryotic cells is the segregation of RNA biogenesis and DNA replication in the nucleus, separate from the cytoplasmic machinery for protein synthesis. As a consequence, messenger RNAs (mRNAs) and all cytoplasmic RNAs from nuclear origin need to be transported from their site of synthesis in the nucleus to their final cytoplasmic destination. Nuclear export occurs through nuclear pore complexes (NPCs) and is mediated by saturable transport receptors, which shuttle between the nucleus and cytoplasm. The past years have seen great progress in the characterization of the mRNA export pathway and the identification of proteins involved in this process. A novel family of nuclear export receptors (the NXF family), distinct from the well-characterized family of importin β-like proteins, has been implicated in the export of mRNA to the cytoplasm. Received 23 January 2001; received after revision 12 April 2001; accepted 12 April 2001     

Transport and diffusion of Tau protein in neurons

In highly polarized and elongated cells such as neurons, Tau protein must enter and move down the axon to fulfill its biological task of stabilizing axonal microtubules. Therefore, cellular systems for distributing Tau molecules are needed. This review discusses different mechanisms that have been proposed to contribute to the dispersion of Tau molecules in neurons. They include (1) directed transport along microtubules as cargo of tubulin complexes and/or motor proteins, (2) diffusion, either through the cytosolic space or along microtubules, and (3) mRNA-based mechanisms such as transport of Tau mRNA into axons and local translation. Diffusion along the microtubule lattice or through the cytosol appear to be the major mechanisms for axonal distribution of Tau protein in the short-to-intermediate range over distances of up to a millimetre. The high diffusion coefficients ensure that Tau can distribute evenly throughout the axonal volume as well as along microtubules. Motor protein-dependent transport of Tau dominates over longer distances and time scales. At low near-physiological levels, Tau is co-transported along with short microtubules from cell bodies into axons by cytoplasmic dynein and kinesin family members at rates of slow axonal transport.     

The bacterial translocase: a dynamic protein channel complex

The major route of protein translocation in bacteria is the so-called general secretion pathway (Sec-pathway). This route has been extensively studied in Escherichia coli and other bacteria. The movement of preproteins across the cytoplasmic membrane is mediated by a multimeric membrane protein complex called translocase. The core of the translocase consists of a proteinaceous channel formed by an oligomeric assembly of the heterotrimeric membrane protein complex SecYEG and the peripheral adenosine triphosphatase (ATPase) SecA as molecular motor. Many secretory proteins utilize the molecular chaperone SecB for targeting and stabilization of the unfolded state prior to translocation, while most nascent inner membrane proteins are targeted to the translocase by the signal recognition particle and its membrane receptor. Translocation is driven by ATP hydrolysis and the proton motive force. In the last decade, genetic and biochemical studies have provided detailed insights into the mechanism of preprotein translocation. Recent crystallographic studies on SecA, SecB and the SecYEG complex now provide knowledge about the structural features of the translocation process. Here, we will discuss the mechanistic and structural basis of the translocation of proteins across and the integration of membrane proteins into the cytoplasmic membrane.Received 10 January 2003; received after revision 2 April 2003; accepted 4 April 2003     

Biogenesis of bacterial inner-membrane proteins

All cells must traffic proteins into and across their membranes. In bacteria, several pathways have evolved to enable protein transfer across the inner membrane, the periplasm, and the outer membrane. The major route of protein translocation in and across the cytoplasmic membrane is the general secretion pathway (Sec-pathway). The biogenesis of membrane proteins not only requires protein translocation but also coordinated targeting to the membrane beforehand and folding and assembly into their protein complexes afterwards to function properly in the cell. All these processes are responsible for the biogenesis of membrane proteins that mediate essential functions of the cell such as selective transport, energy conversion, cell division, extracellular signal sensing, and motility. This review will highlight the most recent developments on the structure and function of bacterial membrane proteins, focusing on the journey that integral membrane proteins take to find their final destination in the inner membrane.     

KIF1Bβ transports dendritically localized mRNPs in neurons and is recruited to synapses in an activity-dependent manner

KIF1Bβ is a kinesin-like, microtubule-based molecular motor protein involved in anterograde axonal vesicular transport in vertebrate and invertebrate neurons. Certain KIF1Bβ isoforms have been implicated in different forms of human neurodegenerative disease, with characterization of their functional integration and regulation in the context of synaptic signaling still ongoing. Here, we characterize human KIF1Bβ (isoform NM015074), whose expression we show to be developmentally regulated and elevated in cortical areas of the CNS (including the motor cortex), in the hippocampus, and in spinal motor neurons. KIF1Bβ localizes to the cell body, axon, and dendrites, overlapping with synaptic-vesicle and postsynaptic-density structures. Correspondingly, in purified cortical synaptoneurosomes, KIF1Bβ is enriched in both pre- and postsynaptic structures, forming detergent-resistant complexes. Interestingly, KIF1Bβ forms RNA–protein complexes, containing the dendritically localized Arc and Calmodulin mRNAs, proteins previously shown to be part of RNA transport granules such as Purα, FMRP and FXR2P, and motor protein KIF3A, as well as Calmodulin. The interaction between KIF1Bβ and Calmodulin is Ca⁺²-dependent and takes place through a domain mapped at the carboxy-terminal tail of the motor. Live imaging of cortical neurons reveals active movement by KIF1Bβ at dendritic processes, suggesting that it mediates the transport of dendritically localized mRNAs. Finally, we show that synaptic recruitment of KIF1Bβ is activity-dependent and increased by stimulation of metabotropic or ionotropic glutamate receptors. The activity-dependent synaptic recruitment of KIF1Bβ, its interaction with Ca²⁺ sensor Calmodulin, and its new role as a dendritic motor of ribonucleoprotein complexes provide a novel basis for understanding the concerted co-ordination of motor protein mobilization and synaptic signaling pathways.     

SecA,a remarkable nanomachine

Biological cells harbor a variety of molecular machines that carry out mechanical work at the nanoscale. One of these nanomachines is the bacterial motor protein SecA which translocates secretory proteins through the protein-conducting membrane channel SecYEG. SecA converts chemically stored energy in the form of ATP into a mechanical force to drive polypeptide transport through SecYEG and across the cytoplasmic membrane. In order to accommodate a translocating polypeptide chain and to release transmembrane segments of membrane proteins into the lipid bilayer, SecYEG needs to open its central channel and the lateral gate. Recent crystal structures provide a detailed insight into the rearrangements required for channel opening. Here, we review our current understanding of the mode of operation of the SecA motor protein in concert with the dynamic SecYEG channel. We conclude with a new model for SecA-mediated protein translocation that unifies previous conflicting data.     

Revisiting the role of mitochondria in spinal muscular atrophy

Cellular and Molecular Life Sciences - Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease of variable clinical severity that is caused by mutations in the survival motor...     

Such small hands: the roles of centrins/caltractins in the centriole and in genome maintenance

Centrins are small, highly conserved members of the EF-hand superfamily of calcium-binding proteins that are found throughout eukaryotes. They play a major role in ensuring the duplication and appropriate functioning of the ciliary basal bodies in ciliated cells. They have also been localised to the centrosome, which is the major microtubule organising centre in animal somatic cells. We describe the identification, cloning and characterisation of centrins in multiple eukaryotic species. Although centrins have been implicated in centriole biogenesis, recent results have indicated that centrosome duplication can, in fact, occur in the absence of centrins. We discuss these data and the non-centrosomal functions that are emerging for the centrins. In particular, we discuss the involvement of centrins in nucleotide excision repair, a process that repairs the DNA lesions that are induced primarily by ultraviolet irradiation. We discuss how centrin may be involved in these diverse processes and contribute to nuclear and cytoplasmic events.     

Processing of heparanase is mediated by syndecan-1 cytoplasmic domain and involves syntenin and α-actinin

Heparanase activity plays a decisive role in cell dissemination associated with cancer metastasis. Cellular uptake of heparanase is considered a pre-requisite for the delivery of latent 65-kDa heparanase to lysosomes and its subsequent proteolytic processing and activation into 8- and 50-kDa protein subunits by cathepsin L. Heparan sulfate proteoglycans, and particularly syndecan, are instrumental for heparanase uptake and activation, through a process that has been shown to occur independent of rafts. Nevertheless, the molecular mechanism underlying syndecan-mediated internalization outside of rafts is unclear. Here, we examined the role of syndecan-1 cytoplasmic domain in heparanase processing, utilizing deletion constructs lacking the entire cytoplasmic domain (Delta), the conserved (C1 or C2), or variable (V) regions. Heparanase processing was markedly increased following syndecan-1 over-expression; in contrast, heparanase was retained at the cell membrane and its processing was impaired in cells over-expressing syndecan-1 deleted for the entire cytoplasmic tail. We have next revealed that conserved domain 2 (C2) and variable (V) regions of syndecan-1 cytoplasmic tail mediate heparanase processing. Furthermore, we found that syntenin, known to interact with syndecan C2 domain, and α actinin are essential for heparanase processing.     

The role of bystin in embryo implantation and in ribosomal biogenesis

Human bystin was identified as a cytoplasmic protein directly binding to trophinin, a cell adhesion molecule potentially involved in human embryo implantation. Although the trophinin gene is unique to mammals, the bystin gene (BYSL) is conserved across eukaryotes. Recent studies show that bystin plays a key role during the transition from silent trophectoderm to an active trophoblast upon trophinin-mediated cell adhesion. Bystin gene knockout and knockdown experiments demonstrate that bystin is essential for embryonic stem cell survival and trophectoderm development in the mouse. Furthermore, biochemical analysis of bystin in human cancer cells and mouse embryos indicates a function in ribosomal biogenesis, specifically in processing of 18S RNA in the 40S subunit. Strong evidence that BYSL is a target of c-MYC is consistent with a role for bystin in rapid protein synthesis, which is required for actively growing cells. Received 30 June 2007; received after revision 7 August 2007; accepted 29 August 2007