Kelin Ru

Long noncoding RNAs in mouse embryonic stem cell pluripotency and differentiation

The transcriptional networks that regulate embryonic stem (ES) cell pluripotency and lineage specification are the subject of considerable attention. To date such studies have focused almost exclusively on protein-coding transcripts. However, recent transcriptome analyses show that the mammalian genome contains thousands of long noncoding RNAs (ncRNAs), many of which appear to be expressed in a developmentally regulated manner. The functions of these remain untested. To identify ncRNAs involved in ES cell biology, we used a custom-designed microarray to examine the expression profiles of mouse ES cells differentiating as embryoid bodies (EBs) over a 16-d time course. We identified 945 ncRNAs expressed during EB differentiation, of which 174 were differentially expressed, many correlating with pluripotency or specific differentiation events. Candidate ncRNAs were identified for further characterization by an integrated examination of expression profiles, genomic context, chromatin state, and promoter analysis. Many ncRNAs showed coordinated expression with genomically associated developmental genes, such as Dlx1, Dlx4, Gata6, and Ecsit. We examined two novel developmentally regulated ncRNAs, Evx1as and Hoxb5/6as, which are derived from homeotic loci and share similar expression patterns and localization in mouse embryos with their associated protein-coding genes. Using chromatin immunoprecipitation, we provide evidence that both ncRNAs are associated with trimethylated H3K4 histones and histone methyltransferase MLL1, suggesting a role in epigenetic regulation of homeotic loci during ES cell differentiation. Taken together, our data indicate that long ncRNAs are likely to be important in processes directing pluripotency and alternative differentiation programs, in some cases through engagement of the epigenetic machinery.

A De Novo Mutation in the β-Tubulin Gene TUBB4A Results in the Leukoencephalopathy Hypomyelination with Atrophy of the Basal Ganglia and Cerebellum

Hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) is a rare hereditary leukoencephalopathy that was originally identified by MRI pattern analysis, and it has thus far defied all attempts at identifying the causal mutation. Only 22 cases are published in the literature to date. We performed exome sequencing on five family trios, two family quartets, and three single probands, which revealed that all eleven H-ABC-diagnosed individuals carry the same de novo single-nucleotide TUBB4A mutation resulting in nonsynonymous change p.Asp249Asn. Detailed investigation of one of the family quartets with the singular finding of an H-ABC-affected sibling pair revealed maternal mosaicism for the mutation, suggesting that rare de novo mutations that are initially phenotypically neutral in a mosaic individual can be disease causing in the subsequent generation. Modeling of TUBB4A shows that the mutation creates a nonsynonymous change at a highly conserved asparagine that sits at the intradimer interface of α-tubulin and β-tubulin, and this change might affect tubulin dimerization, microtubule polymerization, or microtubule stability. Consistent with H-ABCs clinical presentation, TUBB4A is highly expressed in neurons, and a recent report has shown that an N-terminal alteration is associated with a heritable dystonia. Together, these data demonstrate that a single de novo mutation in TUBB4A results in H-ABC.

Mutations in DARS Cause Hypomyelination with Brain Stem and Spinal Cord Involvement and Leg Spasticity

Inherited white-matter disorders are a broad class of diseases for which treatment and classification are both challenging. Indeed, nearly half of the children presenting with a leukoencephalopathy remain without a specific diagnosis. Here, we report on the application of high-throughput genome and exome sequencing to a cohort of ten individuals with a leukoencephalopathy of unknown etiology and clinically characterized by hypomyelination with brain stem and spinal cord involvement and leg spasticity (HBSL), as well as the identification of compound-heterozygous and homozygous mutations in cytoplasmic aspartyl-tRNA synthetase (DARS). These mutations cause nonsynonymous changes to seven highly conserved amino acids, five of which are unchanged between yeast and man, in the DARS C-terminal lobe adjacent to, or within, the active-site pocket. Intriguingly, HBSL bears a striking resemblance to leukoencephalopathy with brain stem and spinal cord involvement and elevated lactate (LBSL), which is caused by mutations in the mitochondria-specific DARS2, suggesting that these two diseases might share a common underlying molecular pathology. These findings add to the growing body of evidence that mutations in tRNA synthetases can cause a broad range of neurologic disorders.

CLONING AND EXPRESSION ANALYSIS OF A NOVEL MOUSE GENE WITH SEQUENCE SIMILARITY TO THE DROSOPHILA FAT FACETS GENE

The Drosophila fat facets (faf) gene is a ubiquitin-specific protease necessary for the normal development of the eye and of the syncytial stage embryo in the fly. Using a gene trap approach in embryonic stem cells we have isolated a murine gene with extensive sequence similarity to the Drosophila faf gene and called it Fam (fat facets in mouse). The putative mouse protein shows colinearity and a high degree of sequence identity to the Drosophila protein over almost its entire length of 2554 amino acids. The two enzymatic sites characteristic of ubiquitin-specific proteases are very highly conserved between mice and Drosophila and this conservation extends to yeast. Fam is expressed in a complex pattern during postimplantation development. In situ hybridisation detected Fam transcripts in the rapidly expanding cell populations of gastrulating and neurulating embryos, in post-mitotic cells of the CNS as well as in the apoptotic regions between the digits, indicating that it is not associated with a single developmental or cellular event. The strong sequence similarity to faf and the developmentally regulated expression pattern suggest that Fam and the ubiquitin pathway may play a role in determining cell fate in mammals, as has been established for Drosophila.

Characterization of G3BPs: tissue specific expression, chromosomal localisation and rasGAP(120) binding studies.

The G3BP (ras‐GTPase‐Activating Protein SH3‐Domain‐Binding Protein) family of proteins has been implicated in both signal transduction and RNA‐metabolism. We have previously identified human G3BP‐1, G3BP‐2, and mouse G3BP‐2. Here, we report the cloning of mouse G3BP‐1, the discovery of two alternatively spliced isoforms of mouse, and human G3BP‐2 (G3BP‐2a and G3BP‐2b), and the chromosomal localisation of human G3BP‐1 and G3BP‐2, which map to 5q14.2‐5q33.3 and 4q12‐4q24 respectively. We mapped the rasGAP120 interactive region of the G3BP‐2 isoforms and show that both G3BP‐2a and G3BP‐2b use an N‐terminal NTF2‐like domain for rasGAP120 binding rather than several available proline‐rich (PxxP) motifs found in members of the G3BPs. Furthermore, we have characterized the protein expression of both G3BP‐1 and G3BP‐2a/b in adult mouse tissues, and show them to be both tissue and isoform specific. J. Cell. Biochem. 84: 173–187, 2002.

Whole exome sequencing in patients with white matter abnormalities

Here we report whole exome sequencing (WES) on a cohort of 71 patients with persistently unresolved white matter abnormalities with a suspected diagnosis of leukodystrophy or genetic leukoencephalopathy. WES analyses were performed on trio, or greater, family groups. Diagnostic pathogenic variants were identified in 35% (25 of 71) of patients. Potentially pathogenic variants were identified in clinically relevant genes in a further 7% (5 of 71) of cases, giving a total yield of clinical diagnoses in 42% of individuals. These findings provide evidence that WES can substantially decrease the number of unresolved white matter cases. Ann Neurol 2016;79:1031–1037

tonB3 Is Required for Normal Twitching Motility and Extracellular Assembly of Type IV Pili

Three mutants with Tn5-B21 insertion in tonB3 (PA0406) of Pseudomonas aeruginosa exhibited defective twitching motility and reduced assembly of extracellular pili. These defects could be complemented with wild-type tonB3.

Isolation and characterization of a new nucleolar protein, Nrap, that is conserved from yeast to humans

Background: The nucleolus is the site of rRNA synthesis and processing in eukaryotic cells, but its composition remains poorly understood.

Detection of aberrant protein phosphorylation in cancer using direct gold-protein affinity interactions

Protein phosphorylation is one of the most prominent post-translational mechanisms for protein regulation, which is frequently impaired in cancer. Through the covalent addition of phosphate groups to certain amino-acids, the interactions of former residues with nearby amino-acids are drastically altered, resulting in major changes of protein conformation that impacts its biological function. Herein, we report that these conformational changes can also disturb the proteins ability to interact with and adsorb onto bare gold surfaces. We exploited this feature to develop a simple electrochemical method for detecting the aberrant phosphorylation of EGFR protein in several lung cancer cell lines. This method, which required as low as 10ng/µL (i.e., 50ng) of purified EGFR protein, also enabled monitoring cell sensitivity to tyrosine kinase inhibitors (TKI) - a common drug used for restoring the function of aberrantly phosphorylated proteins in lung cancer. The reported strategy based on direct gold-protein affinity interactions avoids the conventional paradigm of requiring a phospho-specific antibody for detection and could be a potential alternative of widely used mass spectrometry.

Corrigendum: Mutations in the voltage-gated potassium channel gene KCNH1 cause Temple-Baraitser syndrome and epilepsy.

Corrigendum: Mutations in the voltage-gated potassium channel gene KCNH1 cause Temple-Baraitser syndrome and epilepsy