The role of the RACK1 ortholog Cpc2p in modulating pheromone-induced cell cycle arrest in fission yeast

The detection and amplification of extracellular signals requires the involvement of multiple protein components. In mammalian cells the receptor of activated C kinase (RACK1) is an important scaffolding protein for signal transduction networks. Further, it also performs a critical function in regulating the cell cycle by modulating the G1/S transition. Many eukaryotic cells express RACK1 orthologs, with one example being Cpc2p in the fission yeast Schizosaccharomyces pombe. In contrast to RACK1, Cpc2p has been described to positively regulate, at the ribosomal level, cells entry into M phase. In addition, Cpc2p controls the stress response pathways through an interaction with Msa2p, and sexual development by modulating Ran1p/Pat1p. Here we describe investigations into the role, which Cpc2p performs in controlling the G protein-mediated mating response pathway. Despite structural similarity to Gβ-like subunits, Cpc2p appears not to function at the G protein level. However, upon pheromone stimulation, cells overexpressing Cpc2p display substantial cell morphology defects, disorientation of septum formation and a significantly protracted G1 arrest. Cpc2p has the potential to function at multiple positions within the pheromone response pathway. We provide a mechanistic interpretation of this novel data by linking Cpc2p function, during the mating response, with its previous described interactions with Ran1p/Pat1p. We suggest that overexpressing Cpc2p prolongs the stimulated state of pheromone-induced cells by increasing ste11 gene expression. These data indicate that Cpc2p regulates the pheromone-induced cell cycle arrest in fission yeast by delaying cells entry into S phase

Rationale and design of the plate or pin (pop) study for dislocated midshaft clavicular fractures: study protocol for a randomised controlled trial

To describe the rationale and design of a future study comparing results of plate fixation and Elastic Stable Intramedullary Nailing (ESIN) with a Titanium Elastic Nail (TEN) for adults with a dislocated midshaft clavicular fracture. Prospective randomized multicenter clinical trial in two level 1 and one level 2 trauma centers. 120 patients between 18 and 65 years of age will be included. They are randomized to either plate fixation or ESIN with a TEN with a one year follow-up. Sixty patients will be treated with plate fixation and 60 patients will be treated with ESIN. Primary outcome parameter is the Disabilities of the Arm, Shoulder and Hand score after 6 months. Secondary outcome parameters are Constant Shoulder Score, complications, experienced pain, radiologic consolidation and cosmetics after both procedures. Prospective randomized studies comparing operative techniques for treatment of dislocated midshaft clavicular fracture are lacking. By studying shoulder function, complications, quality of life, radiographic union, cosmetics as well as experienced pain, a complete efficacy assessment of both procedures will be performed. The POP study is registered in the Dutch Trial Register (NTR NTR2438

Large-Scale Evidence for Conservation of NMD Candidature Across Mammals

BACKGROUND: Alternatively-spliced (AS) forms can vary protein function, intracellular localization and post-translational modifications. AS coupled with mRNA nonsense-mediated decay (NMD) can also control the transcript abundance. Here, we have investigated the genome-scale conservation of alternatively-spliced NMD candidates (AS-NMD candidates), in mammals. METHODOLOGY/PRINCIPAL FINDINGS: We mapped>12 million cDNA/EST library transcripts, comprising pooled data from both older and next-generation sequencing techniques, against genomic sequences to annotate AS-NMD candidates generated by in-frame premature termination codons (PTCs), in the human, mouse, rat and cow genomes. In these genomes, we found populations of genes that harbour AS-NMD candidates, varying in number from approximately 149 to 2,051 genes. We discovered that a highly-significant proportion (27%-35%) of AS-NMD candidate genes in mouse, rat and cow, also have human orthologs targeted for NMD. Intron retention was the most abundant type of AS-NMD, ranging from 43% to 67% of genes harbouring an AS-NMD candidate. Groupings of AS-NMD candidate genes either with or without intron retentions also have highly significant AS-NMD conservation, indicating that the trend is not due primarily to conservation of intron retentions. As a subset, the AS-NMD intron retentions are distinguished from non-retained introns by higher GC content, and codon usage similar to the usage in protein-coding sequences. This indicates that most of these alternatively spliced sequences have coded for proteins in the recent evolutionary past. In general, the AS-NMD candidate genes showed a similar pattern of Gene Ontology functional category enrichments in all four species. Genes linked to nucleic-acid interaction and apoptosis, and involved in pathways linked with cancer, were the most common. Finally, we mapped the AS-NMD candidates to mass spectrometry-derived proteomics data, and gathered evidence of truncated polypeptides for at least 10% of all human AS-NMD candidate transcripts. CONCLUSIONS/SIGNIFICANCE: In summary, our analysis provides strong statistical evidence for conservation of functional AS-NMD candidature across Mammalia for a large subset of genes. However, because codon usage of AS-NMD intron retentions is similar to the usage in exons, it is difficult to de-couple conservation of AS-NMD-based regulation from conservation for protein-coding ability, for intron retentions

Somatic ‘Soluble’ Adenylyl Cyclase Isoforms Are Unaffected in Sacytm1Lex/Sacytm1Lex ‘Knockout’ Mice

BACKGROUND: Mammalian Soluble adenylyl cyclase (sAC, Adcy10, or Sacy) represents a source of the second messenger cAMP distinct from the widely studied, G protein-regulated transmembrane adenylyl cyclases. Genetic deletion of the second through fourth coding exons in Sacy(tm1Lex)/Sacy(tm1Lex) knockout mice results in a male sterile phenotype. The absence of any major somatic phenotype is inconsistent with the variety of somatic functions identified for sAC using pharmacological inhibitors and RNA interference. PRINCIPAL FINDINGS: We now use immunological and molecular biological methods to demonstrate that somatic tissues express a previously unknown isoform of sAC, which utilizes a unique start site, and which 'escapes' the design of the Sacy(tm1Lex) knockout allele. CONCLUSIONS/SIGNIFICANCE: These studies reveal increased complexity at the sAC locus, and they suggest that the known isoforms of sAC play a unique function in male germ cells

Wdr74 Is Required for Blastocyst Formation in the Mouse

Preimplantation is a dynamic developmental period during which a combination of maternal and zygotic factors program the early embryo resulting in lineage specification and implantation. A reverse genetic RNAi screen in mouse embryos identified the WD Repeat Domain 74 gene (Wdr74) as being required for these critical first steps of mammalian development. Knockdown of Wdr74 results in embryos that develop normally until the morula stage but fail to form blastocysts or properly specify the inner cell mass and trophectoderm. In Wdr74-deficient embryos, we find activated Trp53-dependent apoptosis as well as a global reduction of RNA polymerase I, II and III transcripts. In Wdr74-deficient embryos blocking Trp53 function rescues blastocyst formation and lineage differentiation. These results indicate that Wdr74 is required for RNA transcription, processing and/or stability during preimplantation development and is an essential gene in the mouse

Malunion after midshaft clavicle fractures in adults: The current view on clavicular malunion in the literature

This is an overview of the current literature on malunion after midshaft clavicle fracture. Anatomy, trauma mechanism, classification, incidence, symptoms, prevention, and treatment options are all discussed. The conclusion is that clavicle malunion is a distinct clinical entity that can be treated successfully

Spag16, an Axonemal Central Apparatus Gene, Encodes a Male Germ Cell Nuclear Speckle Protein that Regulates SPAG16 mRNA Expression

Spag16 is the murine orthologue of Chlamydomonas reinhardtii PF20, a protein known to be essential to the structure and function of the “9+2” axoneme. In Chlamydomonas, the PF20 gene encodes a single protein present in the central pair of the axoneme. Loss of PF20 prevents central pair assembly/integrity and results in flagellar paralysis. Here we demonstrate that the murine Spag16 gene encodes two proteins: 71 kDa SPAG16L, which is found in all murine cells with motile cilia or flagella, and 35 kDa SPAG16S, representing the C terminus of SPAG16L, which is expressed only in male germ cells, and is predominantly found in specific regions within the nucleus that also contain SC35, a known marker of nuclear speckles enriched in pre-mRNA splicing factors. SPAG16S expression precedes expression of SPAG16L. Mice homozygous for a knockout of SPAG16L alone are infertile, but show no abnormalities in spermatogenesis. Mice chimeric for a mutation deleting the transcripts for both SPAG16L and SPAG16S have a profound defect in spermatogenesis. We show here that transduction of SPAG16S into cultured dispersed mouse male germ cells and BEAS-2B human bronchial epithelial cells increases SPAG16L expression, but has no effect on the expression of several other axoneme components. We also demonstrate that the Spag16L promoter shows increased activity in the presence of SPAG16S. The distinct nuclear localization of SPAG16S and its ability to modulate Spag16L mRNA expression suggest that SPAG16S plays an important role in the gene expression machinery of male germ cells. This is a unique example of a highly conserved axonemal protein gene that encodes two protein products with different functions

Novel SPG11 mutations in Asian kindreds and disruption of spatacsin function in the zebrafish

Autosomal recessive hereditary spastic paraplegia with thin corpus callosum (HSP-TCC) maps to the SPG11 locus in the majority of cases. Mutations in the KIAA1840 gene, encoding spatacsin, have been shown to underlie SPG11-linked HSP-TCC. The aim of this study was to perform candidate gene analysis in HSP-TCC subjects from Asian families and to characterize disruption of spatacsin function during zebrafish development. Homozygosity mapping and direct sequencing were used to assess the ACCPN, SPG11, and SPG21 loci in four inbred kindreds originating from the Indian subcontinent. Four novel homozygous SPG11 mutations (c.442+1G>A, c.2146C>T, c.3602_3603delAT, and c.4846C>T) were identified, predicting a loss of spatacsin function in each case. To investigate the role of spatacsin during development, we additionally ascertained the complete zebrafish spg11 ortholog by reverse transcriptase PCR and 5′ RACE. Analysis of transcript expression through whole-mount in situ hybridization demonstrated ubiquitous distribution, with highest levels detected in the brain. Morpholino antisense oligonucleotide injection was used to knock down spatacsin function in zebrafish embryos. Examination of spg11 morphant embryos revealed a range of developmental defects and CNS abnormalities, and analysis of axon pathway formation demonstrated an overall perturbation of neuronal differentiation. These data confirm loss of spatacsin as the cause of SPG11-linked HSP-TCC in Asian kindreds, expanding the mutation spectrum recognized in this disorder. This study represents the first investigation in zebrafish addressing the function of a causative gene in autosomal recessive HSP and identifies a critical role for spatacsin during early neural development in vivo

RACK-1 Acts with Rac GTPase Signaling and UNC-115/abLIM in Caenorhabditis elegans Axon Pathfinding and Cell Migration

Migrating cells and growth cones extend lamellipodial and filopodial protrusions that are required for outgrowth and guidance. The mechanisms of cytoskeletal regulation that underlie cell and growth cone migration are of much interest to developmental biologists. Previous studies have shown that the Arp2/3 complex and UNC-115/abLIM act redundantly to mediate growth cone lamellipodia and filopodia formation and axon pathfinding. While much is known about the regulation of Arp2/3, less is known about regulators of UNC-115/abLIM. Here we show that the Caenorhabditis elegans counterpart of the Receptor for Activated C Kinase (RACK-1) interacts physically with the actin-binding protein UNC-115/abLIM and that RACK-1 is required for axon pathfinding. Genetic interactions indicate that RACK-1 acts cell-autonomously in the UNC-115/abLIM pathway in axon pathfinding and lamellipodia and filopodia formation, downstream of the CED-10/Rac GTPase and in parallel to MIG-2/RhoG. Furthermore, we show that RACK-1 is involved in migration of the gonadal distal tip cells and that the signaling pathways involved in this process might be distinct from those involved in axon pathfinding. In sum, these studies pinpoint RACK-1 as a component of a novel signaling pathway involving Rac GTPases and UNC-115/abLIM and suggest that RACK-1 might be involved in the regulation of the actin cytoskeleton and lamellipodia and filopodia formation in migrating cells and growth cones