The CUGBP2 Splicing Factor Regulates an Ensemble of Branchpoints from Perimeter Binding Sites with Implications for Autoregulation

Alternative pre-mRNA splicing adjusts the transcriptional output of the genome by generating related mRNAs from a single primary transcript, thereby expanding protein diversity. A fundamental unanswered question is how splicing factors achieve specificity in the selection of target substrates despite the recognition of information-poor sequence motifs. The CUGBP2 splicing regulator plays a key role in the brain region-specific silencing of the NI exon of the NMDA R1 receptor. However, the sequence motifs utilized by this factor for specific target exon selection and its role in splicing silencing are not understood. Here, we use chemical modification footprinting to map the contact sites of CUGBP2 to GU-rich motifs closely positioned at the boundaries of the branch sites of the NI exon, and we demonstrate a mechanistic role for this specific arrangement of motifs for the regulation of branchpoint formation. General support for a branch site-perimeter–binding model is indicated by the identification of a group of novel target exons with a similar configuration of motifs that are silenced by CUGBP2. These results reveal an autoregulatory role for CUGBP2 as indicated by its direct interaction with functionally significant RNA motifs surrounding the branch sites upstream of exon 6 of the CUGBP2 transcript itself. The perimeter-binding model explains how CUGBP2 can effectively embrace the branch site region to achieve the specificity needed for the selection of exon targets and the fine-tuning of alternative splicing patterns

WRN Mutation Update: Mutation Spectrum, Patient Registries, and Translational Prospects: HUMAN MUTATION

Werner syndrome (WS) is a rare autosomal recessive disorder characterized by a constellation of adult onset phenotypes consistent with an acceleration of intrinsic biological aging. It is caused by pathogenic variants in the WRN gene, which encodes a multifunctional nuclear protein with exonuclease and helicase activities. WRN protein is thought to be involved in optimization of various aspects of DNA metabolism, including DNA repair, recombination, replication, and transcription. In this update, we summarize a total of 83 different WRN mutations, including eight previously unpublished mutations identified by the International Registry of Werner Syndrome (Seattle, WA) and the Japanese Werner Consortium (Chiba, Japan), as well as 75 mutations already reported in the literature. The Seattle International Registry recruits patients from all over the world to investigate genetic causes of a wide variety of progeroid syndromes in order to contribute to the knowledge of basic mechanisms of human aging. Given the unusually high prevalence of WS patients and heterozygous carriers in Japan, the major goal of the Japanese Consortium is to develop effective therapies and to establish management guidelines for WS patients in Japan and elsewhere. This review will also discuss potential translational approaches to this disorder, including those currently under investigation

Genomic Relationships, Novel Loci, and Pleiotropic Mechanisms across Eight Psychiatric Disorders

Genetic influences on psychiatric disorders transcend diagnostic boundaries, suggesting substantial pleiotropy of contributing loci. However, the nature and mechanisms of these pleiotropic effects remain unclear. We performed analyses of 232,964 cases and 494,162 controls from genome-wide studies of anorexia nervosa, attention-deficit/hyper-activity disorder, autism spectrum disorder, bipolar disorder, major depression, obsessive-compulsive disorder, schizophrenia, and Tourette syndrome. Genetic correlation analyses revealed a meaningful structure within the eight disorders, identifying three groups of inter-related disorders. Meta-analysis across these eight disorders detected 109 loci associated with at least two psychiatric disorders, including 23 loci with pleiotropic effects on four or more disorders and 11 loci with antagonistic effects on multiple disorders. The pleiotropic loci are located within genes that show heightened expression in the brain throughout the lifespan, beginning prenatally in the second trimester, and play prominent roles in neurodevelopmental processes. These findings have important implications for psychiatric nosology, drug development, and risk prediction.Peer reviewe

Internet Commerce: Security Is Still A Concern

As the number of organizations that advertise over the Internet continues to grow, they are looking for opportunities to expand their market areas through electronic commerce.  However, the majority of potential users are still concerned with the security of their transactions over the Internet

Rapid sample preparation with Lyse-It® for <i>Listeria monocytogenes</i> and <i>Vibrio cholerae</i>

<div><p>Sample preparation is a leading bottleneck in rapid detection of pathogenic bacteria. Here, we use Lyse-It^® for bacterial cellular lysis, genomic DNA fragmentation, and protein release and degradation for both <i>Listeria monocytogenes</i> and <i>Vibrio cholerae</i>. The concept of Lyse-It^® employs a conventional microwave and Lyse-It^® slides for intensely focused microwave irradiation onto the sample. High microwave power and a <60 second irradiation time allow for rapid cellular lysis and subsequent intracellular component release. The pathogenic bacteria are identified by quantitative polymerase chain reaction (qPCR), which subsequently demonstrates the viability of DNA for amplification post microwave-induced lysis. Intracellular component release, degradation, and detection of <i>L</i>. <i>monocytogenes</i> and <i>V</i>. <i>cholerae</i> has been performed and shown in this paper. These results demonstrate a rapid, low-cost, and efficient way for bacterial sample preparation on both food and water-borne Gram-positive and -negative organisms alike.</p></div

Dynamic Light Scattering (DLS) of microwave lysed <i>V</i>. <i>cholerae</i> and <i>L</i>. <i>monocytogenes</i> with and without Lyse-It^®.

<p><b>(A)</b><i>V</i>. <i>cholerae</i> microwave irradiated for 60 seconds at varying powers and <b>(B)</b> microwaved for varying times at 30% power. <b>(C)</b> <i>L</i>. <i>monocytogenes</i> microwave irradiated for varying powers for 60 seconds and <b>(D)</b> microwaved at 50% power for varying times. It is clear that when Lyse-It^® is used, there is a decrease in the overall cellular size indicating that cellular lysis is occurring and is more efficient with Lyse-It^® as compared to standard microwave heating.</p

Microwave cellular lysis visualization using a mini-gel instrument.

<p>Aligent 2100 Mini-gel Bioanalyzer analysis of <i>L</i>. <i>monocytogenes</i> lysate microwave lysed with Lyse-It^®. As the power and the time of the microwave increases the overall DNA concentration decreases indicating more continues shearing (cutting) of genomic DNA, to smaller and smaller sizes.</p

Microwave cellular lysis visualization through gel electrophoresis.

<p>Microwave cellular lysis of [10⁸ cfu] <i>V</i>. <i>cholerae</i> at 30% power (270 watts), (<b>Left</b>) Standard microwave heating, (<b>Right</b>) Microwave heating with Lyse-It^®. L: 100 bp ladder, 1: Pre <i>V</i>. <i>cholerae</i> microwave irradiation, 2–6: 15–75 seconds no Lyse-It^®, 7: Pre <i>V</i>. <i>cholerae</i> microwave irradiation, 8–12:15–75 seconds Lyse-It^®.</p

Microwave irradiation temperatures.

<p>Microwave irradiation temperatures.</p

SDS PAGE of <i>V</i>. <i>cholerae</i>.

<p><b>(A)</b><i>V</i>. <i>cholerae</i> conventionally heating and microwave irradiated. L: Ladder, P: Pre-Lyse <i>V</i>. <i>cholerae</i>. <b>Left:</b> Conventionally heating. Lanes 1–5: 40°C—80°C, <b>Right:</b> Microwave irradiated. Lanes 1–5 No Lyse-It^® 10–50% power, 60 seconds respectively. Lanes 6–10: Lyse-It^® 10–50% power, 60 seconds respectively. <b>Highlighted regions</b> demonstrate that more protein is released with Lyse-It^®.</p