Amphids in Strongyloides stercoralis and other parasitic nematodes

In this review, Francis Ashton and Gerhard Schad examine the ultrastructure of the amphids of several animal parasitic nematodes. These structures are the main chemosensory organs of these worms and probably play an important role in host-finding behavior and the control of development. Reconstructions made from serial micrographs of the neurons in the amphids of the threadworm Strongyloides stercoralis are shown. These stereo images permit three-dimensional visualization of these complex sense organs. The association between each amphidial neuron and its cell body has not been made previously for a parasitic nematode; however, this has been done for the free-living nematode Caenorhabditis elegans, which served as a model for these studies. Recognition of the cell bodies will provide a point of departure for laser microbeam ablation studies to determine individual neuronal function.     

NMR reveals novel mechanisms of protein activity regulation

NMR spectroscopy is one of the most powerful tools for the characterization of biomolecular systems. A unique aspect of NMR is its capacity to provide an integrated insight into both the structure and intrinsic dynamics of biomolecules. In addition, NMR can provide site-resolved information about the conformation entropy of binding, as well as about energetically excited conformational states. Recent advances have enabled the application of NMR for the characterization of supramolecular systems. A summary of mechanisms underpinning protein activity regulation revealed by the application of NMR spectroscopy in a number of biological systems studied in the lab is provided.     

Global regulation of alternative splicing during myogenic differentiation

Recent genome-wide analyses have elucidated the extent of alternative splicing (AS) in mammals, often focusing on comparisons of splice isoforms between differentiated tissues. However, regulated splicing changes are likely to be important in biological transitions such as cellular differentiation, or response to environmental stimuli. To assess the extent and significance of AS in myogenesis, we used splicing-sensitive microarray analysis of differentiating C2C12 myoblasts. We identified 95 AS events that undergo robust splicing transitions during C2C12 differentiation. More than half of the splicing transitions are conserved during differentiation of avian myoblasts, suggesting the products and timing of transitions are functionally significant. The majority of splicing transitions during C2C12 differentiation fall into four temporal patterns and were dependent on the myogenic program, suggesting that they are integral components of myogenic differentiation. Computational analyses revealed enrichment of many sequence motifs within the upstream and downstream intronic regions near the alternatively spliced regions corresponding to binding sites of splicing regulators. Western analyses demonstrated that several splicing regulators undergo dynamic changes in nuclear abundance during differentiation. These findings show that within a developmental context, AS is a highly regulated and conserved process, suggesting a major role for AS regulation in myogenic differentiation.     

Molecular phylogenetic analysis of the genus Strongyloides and related nematodes

Strongyloides spp., parasitic nematodes of humans and many other terrestrial vertebrates, display an unusual heterogonic lifecycle involving alternating parasitic and free-living adult reproductive stages. A number of other genera have similar lifecycles, but their relationships to Strongyloides have not been clarified. We have inferred a phylogeny of 12 species of Strongyloides, Parastrongyloides, Rhabdias and Rhabditophanes using small subunit ribosomal RNA gene (SSU rDNA) sequences. The lineage leading to Strongyloides appears to have arisen within parasites of terrestrial invertebrates. Inferred lifecycle evolution was particularly dynamic within these nematodes. Importantly, the free-living Rhabditophanes sp. KR3021 is placed within a clade of parasitic taxa, suggesting that this species may represent a reversion to a non-parasitic lifecycle. Species within the genus Strongyloides are very closely related, despite the disparity of host species parasitised. The highly pathogenic human parasite Strongyloides fuelleborni kelleyi is not supported as a subspecies of the primate parasite S. fuelleborni fuelleborni, but is most likely derived from a local zoonotic source.     

Melanocyte regeneration reveals mechanisms of adult stem cell regulation

Utilization of adult stem cells in regenerative therapies may require a thorough understanding of the mechanisms that establish, recruit and renew the stem cell, promote the differentiation of its daughters, or how the stem cell is repressed by its target tissue. Regeneration of melanocytes in the regenerating zebrafish caudal fin, or following larval melanocyte-specific ablation, or recruitment of new melanocytes during pigment pattern metamorphosis each provides evidence for melanocyte stem cells (MSCs) that support the melanocyte pigment pattern. We discuss the mechanisms of MSC regulation provided from analysis of normal or mutant regeneration in each of these systems, including the implications drawn from evidence that regeneration does not simply recapitulate ontogenetic development. These results suggest that analysis of melanocyte regeneration in zebrafish will provide a fine scale dissection of mechanisms establishing or regulating adult stem cells.     

Constant-ratio alternative substrate approach for differentiation of enzyme mechanisms

A new kinetic approach using alternative substrates as a tool for studying enzyme mechanisms is described. In this method the substrate to alternative substrate ratio is maintained constant and the common product (or summation of product analogs) is measured. The double-reciprocal plots so obtained at several constant ratios generate different patterns for various mechanisms, thus permitting a choice of kinetic model. In some cases, secondary intercept plots are utilized as a diagnostic aid. Another feature of this approach is that most of the resultant plots are linear. The graphical patterns for four cases of two-substrate, two-product reactions are presented as examples. These patterns allow one to differentiate several mechanisms which are not distinguishable by conventional alternative substrate, competitive inhibitor, or product inhibition studies alone. When used in combination with other methods, various mechanisms involving isomerization and abortive complex formation can be differentiated even if only one alternative substrate is available.     

选择性多聚腺苷酸化的生物学效应及其调控机制研究进展

真核生物基因的前体mRNA(pre-mRNA)及一些lncRNA在成熟过程中其3'端会发生剪切和多聚腺苷酸化反应(cleavage and polyadenylation, C/P),C/P的发生需要多聚腺苷酸化信号(polyadenylation signal, PAS)的存在。选择性多聚腺苷酸化(alternative cleavage and polyadenylation, APA)是指具有多个PAS的基因,在其mRNA3'端成熟过程中,由于选择不同的PAS,导致产生出多个3'UTR长度和序列组成不同的转录异构体。3'UTR长度和序列的不同会影响mRNA的稳定性、翻译效率、运输和细胞定位等,因此APA是真核生物的一个重要转录后调控方式。近年来,对大量动物、植物及酵母的基因组测序分析发现,APA在真核生物广泛存在,针对APA的生物学效应和调控机制开展了一系列研究。目前已鉴定出许多APA调控的顺式调控元件和反式作用因子。本文重点介绍了APA生物学效应和调控机制的最新研究进展,并探讨了未来APA调控的研究方向。     

Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins

Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here, we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre-mRNA splicing (AS) in human cells. Using splicing-sensitive microarrays, crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq), and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveal position-dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross- and autoregulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells.     

Structural-functional organization of the adenylyl cyclases in unicellular eukaryotes and molecular mechanisms of its regulation

At the present time, adenylyl cyclases (ACs)--the enzymes, catalyzing the formation of second messenger cAMP, were found in yeasts and related fungi, amoeba Dictyostelium discoideum, flagellates, malaria plasmodium, ciliates. However, structural-functional organization of the ACs and molecular mechanisms of its regulation are different to great extent. The scores of structurally related ACs, one time penetrating the membrane and possessing the receptor function, were identified in flagellates. Three types of ACs, strongly differed in the topology, the domain organization and the sensitivity to regulatory molecules and physical factors, were found in amoeba D. discoideum. One of them (AC-A) is close to membrane-bound ACs of the mammals and can be regulated by extracellular cAMP. It was shown that the enzymes of the yeasts, lacking the transmembrane domains, formed the intermolecular complexes, which were stabilized by the interactions between leucine-rich repeat regions. The data presented in the review give evidence that the main molecular mechanisms of the functioning of vertebrate ACs were formed in unicellular organisms and fungi. At the same time the structure and functions of the ACs of the lower eukaryotes are strongly varied. It can be connected with the special features of life cycle of the lower eukaryotes and with the realization of different models of functioning and regulation of cAMP-dependent cascades at the earlier steps of evolution.     

Genomic organization, proposed alternative splicing mechanisms, and RNA editing structure of GRIK1

In the present study, we report the genomic reconstruction of the glutamate receptor GluR5 gene (GRIK1, alias GLUR5; 402 kb) by the use of the data available in public databases. This analysis allowed characterization of the exon-intron boundaries, identification of the gene promoter region, presentation of the putative complete sequences of two GluR5 mRNA isoforms, and definition of the structure of GluR5 pre-mRNA surrounding the Q/R editing site.     

Trimeric structure of PRL-1 phosphatase reveals an active enzyme conformation and regulation mechanisms

The PRL phosphatases, which constitute a subfamily of the protein tyrosine phosphatases (PTPs), are implicated in oncogenic and metastatic processes. Here, we report the crystal structure of human PRL-1 determined at 2.7A resolution. The crystal structure reveals the shallow active-site pocket with highly hydrophobic character. A structural comparison with the previously determined NMR structure of PRL-3 exhibits significant differences in the active-site region. In the PRL-1 structure, a sulfate ion is bound to the active-site, providing stabilizing interactions to maintain the canonically found active conformation of PTPs, whereas the NMR structure exhibits an open conformation of the active-site. We also found that PRL-1 forms a trimer in the crystal and the trimer exists in the membrane fraction of cells, suggesting the possible biological regulation of PRL-1 activity by oligomerization. The detailed structural information on the active enzyme conformation and regulation of PRL-1 provides the structural basis for the development of potential inhibitors of PRL enzymes.     

大型真菌子实体发生的形态学过程及调控机制

大型真菌中很多种类具有较高的营养价值和药用价值,而食药用部位多为大型真菌的子实体,所以子实体的形成对大型真菌的开发应用就显得尤为重要。在大型真菌生活史中,子实体的发生揭示着真菌完成了从营养生长向生殖生长的转变。从理论上来说,菌丝体只需完成营养生长,即可进入生殖生长;而实际上,子实体的发生受到各种环境因素、遗传因素等的影响。因此,本文从子实体发生的形态学过程、环境影响因素、分子调控机制等方面综述了近年国内外关于大型真菌子实体发生的研究概况,为大型真菌的系统研究和重要食药用菌的栽培驯化提供理论借鉴和参考依据。     

RNA-Seq analysis in mutant zebrafish reveals role of U1C protein in alternative splicing regulation

Precise 5' splice-site recognition is essential for both constitutive and regulated pre-mRNA splicing. The U1 small nuclear ribonucleoprotein particle (snRNP)-specific protein U1C is involved in this first step of spliceosome assembly and important for stabilizing early splicing complexes. We used an embryonically lethal U1C mutant zebrafish, hi1371, to investigate the potential genomewide role of U1C for splicing regulation. U1C mutant embryos contain overall stable, but U1C-deficient U1 snRNPs. Surprisingly, genomewide RNA-Seq analysis of mutant versus wild-type embryos revealed a large set of specific target genes that changed their alternative splicing patterns in the absence of U1C. Injection of ZfU1C cRNA into mutant embryos and in vivo splicing experiments in HeLa cells after siRNA-mediated U1C knockdown confirmed the U1C dependency and specificity, as well as the functional conservation of the effects observed. In addition, sequence motif analysis of the U1C-dependent 5' splice sites uncovered an association with downstream intronic U-rich elements. In sum, our findings provide evidence for a new role of a general snRNP protein, U1C, as a mediator of alternative splicing regulation.     

Structure-functional organization of adenylyl cyclases of unicellular eukaryotes and molecular mechanisms of their regulation

Adenylyl cyclases, the enzymes which catalyze the formation of the second messenger cAMP, are presently known to exist in yeast and related fungi, the amoeba Dictyostelium discoideum, flagellates, plasmodium, and infusoria. However, their structure-functional organization and molecular mechanisms of regulation differ considerably. Thus, in flagellates, tens of structurally similar adenylyl cyclase one-pass transmembrane proteins performing receptor functions have been discovered. In the amoeba D. discoideum, three types of adenylyl cyclases were detected, which differ by their topology, domain organization, and sensitivity to regulatory molecules and physical factors, one of which, adenylyl cyclase-A (AC-A), is similar to mammalian membrane-bound adenylyl cyclases and regulated by extracellular cAMP. Yeasts, in turn, have been shown to possess adenylyl cyclases that do not have transmembrane domains, but are able to form intermolecular complexes stabilized by interactions between repeated regions enriched in leucine residues. The data presented in this review indicate that the main molecular mechanisms underlying the actions of vertebrate adenylyl cyclases evolved as early as in the unicellular organisms and fungi. The structures and functions of adenylyl cyclases of the lower eukaryotes are much more diverse, which might be due both to the peculiarities of their life cycles and to the development at the initial stages of evolution of different models for the functioning and regulation of cAMP-dependent signaling cascades.     

BIM regulates apoptosis during mammary ductal morphogenesis, and its absence reveals alternative cell death mechanisms

The adult, virgin mammary gland is a highly organized tree-like structure formed by ducts with hollowed lumen. Although lumen formation during pubertal development appears to involve apoptosis, the molecular mechanisms that regulate this process are not known. Here, we demonstrate that disruption of the BH3-only proapoptotic factor Bim in mice prevents induction of apoptosis in and clearing of the lumen in terminal end buds during puberty. However, cells that fill the presumptive luminal space are eventually cleared from the adjacent ducts by a caspase-independent death process. Within the filled Bim(-/-) ducts, epithelial cells are deprived of matrix attachment and undergo squamous differentiation prior to clearing. Similarly, we also detect squamous differentiation in vitro when immortalized mammary epithelial cells are detached from the matrix. These data provide important mechanistic information on the processes involved in sculpting the mammary gland and demonstrate that BIM is a critical regulator of apoptosis in vivo.     

Mathematical modeling reveals the mechanisms of feedforward regulation in cell fate decisions in budding yeast

The determination of cell fate is one of the key questions of developmental biology. Recent experiments showed that feedforward regulation is a novel feature of regulatory networks that controls reversible cellular transitions. However, the underlying mechanism of feedforward regulation-mediated cell fate decision is still unclear. Therefore, using experimental data, we develop a full mathematical model of the molecular network responsible for cell fate selection in budding yeast. To validate our theoretical model, we first investigate the dynamical behaviors of key proteins at the Start transition point and the G1/S transition point; a crucial three-node motif consisting of cyclin (Cln1/2), Substrate/Subunit Inhibitor of cyclin-dependent protein kinase (Sic1) and cyclin B (Clb5/6) is considered at these points. The rapid switches of these important components between high and low levels at two transition check points are demonstrated reasonably by our model. Many experimental observations about cell fate decision and cell size control are also theoretically reproduced. Interestingly, the feedforward regulation provides a reliable separation between different cell fates. Next, our model reveals that the threshold for the amount of WHIskey (Whi5) removed from the nucleus is higher at the Reentry point in pheromone-arrested cells compared with that at the Start point in cycling cells. Furthermore, we analyze the hysteresis in the cell cycle kinetics in response to changes in pheromone concentration, showing that Cln3 is the primary driver of reentry and Cln1/2 is the secondary driver of reentry. In particular, we demonstrate that the inhibition of Cln1/2 due to the accumulation of Factor ARrest (Far1) directly reinforces arrest. Finally, theoretical work verifies that the three-node coherent feedforward motif created by cell FUSion (Fus3), Far1 and STErile (Ste12) ensures the rapid arrest and reversibility of a cellular state. The combination of our theoretical model and the previous experimental data contributes to the understanding of the molecular mechanisms of the cell fate decision at the G1 phase in budding yeast and will stimulate further biological experiments in future.