Deficiency in the endocytic adaptor proteins PHETA1/2 impairs renal and craniofacial development

A critical barrier in the treatment of endosomal and lysosomal diseases is the lack of understanding of the in vivo functions of the putative causative genes. We addressed this by investigating a key pair of endocytic adaptor proteins, PH domain-containing endocytic trafficking adaptor 1 and 2 (PHETA1/2; also known as FAM109A/B, Ses1/2, IPIP27A/B), which interact with the protein product of OCRL, the causative gene for Lowe syndrome. Here, we conducted the first study of PHETA1/2 in vivo, utilizing the zebrafish system. We found that impairment of both zebrafish orthologs, pheta1 and pheta2, disrupted endocytosis and ciliogenesis in renal tissues. In addition, pheta1/2 mutant animals exhibited reduced jaw size and delayed chondrocyte differentiation, indicating a role in craniofacial development. Deficiency of pheta1/2 resulted in dysregulation of cathepsin K, which led to an increased abundance of type II collagen in craniofacial cartilages, a marker of immature cartilage extracellular matrix. Cathepsin K inhibition rescued the craniofacial phenotypes in the pheta1/2 double mutants. The abnormal renal and craniofacial phenotypes in the pheta1/2 mutant animals were consistent with the clinical presentation of a patient with a de novo arginine (R) to cysteine (C) variant (R6C) of PHETA1. Expressing the patient-specific variant in zebrafish exacerbated craniofacial deficits, suggesting that the R6C allele acts in a dominant-negative manner. Together, these results provide insights into the in vivo roles of PHETA1/2 and suggest that the R6C variant is contributory to the pathogenesis of disease in the patient

Action Experience and Action Discovery in Medicated Individuals with Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder that markedly affects voluntary action. While regular dopamine treatment can help restore motor function, dopamine also influences cognitive portions of the action system. Previous studies have demonstrated that dopamine medication boosts action-effect associations, which are crucial for the discovery of new voluntary actions. In the present study, we investigated whether neural processes involved in the discovery of new actions are altered in PD participants on regular dopamine treatment, compared to healthy age-matched controls. We recorded brain electroencephalography (EEG) activity while PD patients and age-matched controls performed action discovery (AD) and action control tasks. We found that the novelty P3, a component normally present when there is uncertainty about the occurrence of the sensory effect, was enhanced in PD patients. However, AD was maintained in PD patients, and the novelty P3 demonstrated normal learning-related reductions. Crucially, we found that in PD patients the causal association between an action and its resulting sensory outcome did not modulate the amplitude of the feedback correct-related positivity (fCRP), an EEG component sensitive to the association between an action and its resulting effect. Collectively, these preliminary results suggest that the formation of long-term action-outcome representations may be maintained in PD patients on regular dopamine treatment, but the initial experience of action-effect association may be affected

RNA Modulators of Complex Phenotypes in Mammalian Cells

RNA-mediated gene silencing, in the form of RNA interference (RNAi) or microRNAs (miRNAs) has provided novel tools for gene discovery and validation in mammalian cells. Here, we report on the construction and application of a random small RNA expression library for use in identifying small interfering RNA (siRNA) effectors that can modify complex cellular phenotypes in mammalian cells. The library is based in a retroviral vector and uses convergent promoters to produce unique small complementary RNAs. Using this library, we identify a range of small RNA-encoding gene inserts that overcome resistance to 5-fluorouracil (5-FU)- or tumour necrosis factor alpha (TNF-α)- induced cell death in colorectal cancer cells. We demonstrate the utility of this technology platform by identifying a key RNA effector, in the form of a siRNA, which overcomes cell death induced by the chemotherapeutic 5-FU. The technology described has the potential to identify both functional RNA modulators capable of altering physiological systems and the cellular target genes altered by these modulators

Implicit Temporal Expectation Attenuates Auditory Attentional Blink

Attentional blink (AB) describes a phenomenon whereby correct identification of a first target impairs the processing of a second target (i.e., probe) nearby in time. Evidence suggests that explicit attention orienting in the time domain can attenuate the AB. Here, we used scalp-recorded, event-related potentials to examine whether auditory AB is also sensitive to implicit temporal attention orienting. Expectations were set up implicitly by varying the probability (i.e., 80% or 20%) that the probe would occur at the +2 or +8 position following target presentation. Participants showed a significant AB, which was reduced with the increased probe probability at the +2 position. The probe probability effect was paralleled by an increase in P3b amplitude elicited by the probe. The results suggest that implicit temporal attention orienting can facilitate short-term consolidation of the probe and attenuate auditory AB

Crystal Structure of TDRD3 and Methyl-Arginine Binding Characterization of TDRD3, SMN and SPF30

SMN (Survival motor neuron protein) was characterized as a dimethyl-arginine binding protein over ten years ago. TDRD3 (Tudor domain-containing protein 3) and SPF30 (Splicing factor 30 kDa) were found to bind to various methyl-arginine proteins including Sm proteins as well later on. Recently, TDRD3 was shown to be a transcriptional coactivator, and its transcriptional activity is dependent on its ability to bind arginine-methylated histone marks. In this study, we systematically characterized the binding specificity and affinity of the Tudor domains of these three proteins quantitatively. Our results show that TDRD3 preferentially recognizes asymmetrical dimethylated arginine mark, and SMN is a very promiscuous effector molecule, which recognizes different arginine containing sequence motifs and preferentially binds symmetrical dimethylated arginine. SPF30 is the weakest methyl-arginine binder, which only binds the GAR motif sequences in our library. In addition, we also reported high-resolution crystal structures of the Tudor domain of TDRD3 in complex with two small molecules, which occupy the aromatic cage of TDRD3

An RNA Transport System in Candida albicans Regulates Hyphal Morphology and Invasive Growth

Localization of specific mRNAs is an important mechanism through which cells achieve polarity and direct asymmetric growth. Based on a framework established in Saccharomyces cerevisiae, we describe a She3-dependent RNA transport system in Candida albicans, a fungal pathogen of humans that grows as both budding (yeast) and filamentous (hyphal and pseudohyphal) forms. We identify a set of 40 mRNAs that are selectively transported to the buds of yeast-form cells and to the tips of hyphae, and we show that many of the genes encoded by these mRNAs contribute to hyphal development, as does the transport system itself. Although the basic system of mRNA transport is conserved between S. cerevisiae and C. albicans, we find that the cargo mRNAs have diverged considerably, implying that specific mRNAs can easily move in and out of transport control over evolutionary timescales. The differences in mRNA cargos likely reflect the distinct selective pressures acting on the two species

An efficient genetic algorithm for structural RNA pairwise alignment and its application to non-coding RNA discovery in yeast

<p>Abstract</p> <p>Background</p> <p>Aligning RNA sequences with low sequence identity has been a challenging problem since such a computation essentially needs an algorithm with high complexities for taking structural conservation into account. Although many sophisticated algorithms for the purpose have been proposed to date, further improvement in efficiency is necessary to accelerate its large-scale applications including non-coding RNA (ncRNA) discovery.</p> <p>Results</p> <p>We developed a new genetic algorithm, Cofolga2, for simultaneously computing pairwise RNA sequence alignment and consensus folding, and benchmarked it using BRAliBase 2.1. The benchmark results showed that our new algorithm is accurate and efficient in both time and memory usage. Then, combining with the originally trained SVM, we applied the new algorithm to novel ncRNA discovery where we compared <it>S. cerevisiae </it>genome with six related genomes in a pairwise manner. By focusing our search to the relatively short regions (50 bp to 2,000 bp) sandwiched by conserved sequences, we successfully predict 714 intergenic and 1,311 sense or antisense ncRNA candidates, which were found in the pairwise alignments with stable consensus secondary structure and low sequence identity (≤ 50%). By comparing with the previous predictions, we found that > 92% of the candidates is novel candidates. The estimated rate of false positives in the predicted candidates is 51%. Twenty-five percent of the intergenic candidates has supports for expression in cell, i.e. their genomic positions overlap those of the experimentally determined transcripts in literature. By manual inspection of the results, moreover, we obtained four multiple alignments with low sequence identity which reveal consensus structures shared by three species/sequences.</p> <p>Conclusion</p> <p>The present method gives an efficient tool complementary to sequence-alignment-based ncRNA finders.</p

A Cytoplasmic Complex Mediates Specific mRNA Recognition and Localization in Yeast

The localization of ash mRNA in yeast requires the binding of She2p and the myosin adaptor protein She3p to its localization element, which is highly specific and leads to the assembly of stable transport complexes

Remote control of gene function by local translation

The subcellular position of a protein is a key determinant of its function. Mounting evidence indicates that RNA localization, where specific mRNAs are transported subcellularly and subsequently translated in response to localized signals, is an evolutionarily conserved mechanism to control protein localization. On-site synthesis confers novel signaling properties to a protein and helps to maintain local proteome homeostasis. Local translation plays particularly important roles in distal neuronal compartments, and dysregulated RNA localization and translation cause defects in neuronal wiring and survival. Here, we discuss key findings in this area and possible implications of this adaptable and swift mechanism for spatial control of gene function