NMR Analysis of the Dynamic Exchange of the NS2B Cofactor between Open and Closed Conformations of the West Nile Virus NS2B-NS3 Protease

Dengue and West Nile virus infections put an estimated 2.5 billion people at risk. Neither drugs nor vaccines are currently available against these diseases. The non-structural protein NS3 is a protease that, together with the cofactor NS2B, is essential for viral maturation. The NS2B-NS3 proteases of dengue and West Nile viruses are highly homologous and present promising drug targets. Crystal structures of the West Nile virus protease with and without bound inhibitor revealed large structural differences in NS2B, while no crystal structure of the dengue virus protease could be determined with a bound inhibitor. We investigated the structural change in solution and found that the C-terminal segment (CTS) of the NS2B cofactor is prone to dissociation from NS3. In the case of the West Nile virus protease, the CTS of NS2B is mostly associated with NS3, especially in the presence of inhibitors. In the case of the dengue virus protease and in the absence of inhibitors, the CTS of NS2B is mostly dissociated from NS3. Finding drug candidates to inhibit the association of the NS2B cofactor may thus be easier for the dengue virus protease

The RNA binding protein HuR differentially regulates unique subsets of mRNAs in estrogen receptor negative and estrogen receptor positive breast cancer

<p>Abstract</p> <p>Background</p> <p>The discordance between steady-state levels of mRNAs and protein has been attributed to posttranscriptional control mechanisms affecting mRNA stability and translation. Traditional methods of genome wide microarray analysis, profiling steady-state levels of mRNA, may miss important mRNA targets owing to significant posttranscriptional gene regulation by RNA binding proteins (RBPs).</p> <p>Methods</p> <p>The ribonomic approach, utilizing RNA immunoprecipitation hybridized to microarray (RIP-Chip), provides global identification of putative endogenous mRNA targets of different RBPs. HuR is an RBP that binds to the AU-rich elements (ARE) of labile mRNAs, such as proto-oncogenes, facilitating their translation into protein. HuR has been shown to play a role in cancer progression and elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. HuR has been described to control genes in several of the acquired capabilities of cancer and has been hypothesized to be a tumor-maintenance gene, allowing for cancers to proliferate once they are established.</p> <p>Results</p> <p>We used HuR RIP-Chip as a comprehensive and systematic method to survey breast cancer target genes in both MCF-7 (estrogen receptor positive, ER+) and MDA-MB-231 (estrogen receptor negative, ER-) breast cancer cell lines. We identified unique subsets of HuR-associated mRNAs found individually or in both cell types. Two novel HuR targets, <it>CD9 </it>and <it>CALM2 </it>mRNAs, were identified and validated by quantitative RT-PCR and biotin pull-down analysis.</p> <p>Conclusion</p> <p>This is the first report of a side-by-side genome-wide comparison of HuR-associated targets in wild type ER+ and ER- breast cancer. We found distinct, differentially expressed subsets of cancer related genes in ER+ and ER- breast cancer cell lines, and noted that the differential regulation of two cancer-related genes by HuR was contingent upon the cellular environment.</p

Regulation of MicroRNA Biogenesis: A miRiad of mechanisms

microRNAs are small, non-coding RNAs that influence diverse biological functions through the repression of target genes during normal development and pathological responses. Widespread use of microRNA arrays to profile microRNA expression has indicated that the levels of many microRNAs are altered during development and disease. These findings have prompted a great deal of investigation into the mechanism and function of microRNA-mediated repression. However, the mechanisms which govern the regulation of microRNA biogenesis and activity are just beginning to be uncovered. Following transcription, mature microRNA are generated through a series of coordinated processing events mediated by large protein complexes. It is increasingly clear that microRNA biogenesis does not proceed in a 'one-size-fits-all' manner. Rather, individual classes of microRNAs are differentially regulated through the association of regulatory factors with the core microRNA biogenesis machinery. Here, we review the regulation of microRNA biogenesis and activity, with particular focus on mechanisms of post-transcriptional control. Further understanding of the regulation of microRNA biogenesis and activity will undoubtedly provide important insights into normal development as well as pathological conditions such as cardiovascular disease and cancer

Targeted Next-Generation Sequencing Analysis of 1,000 Individuals with Intellectual Disability.

To identify genetic causes of intellectual disability (ID), we screened a cohort of 986 individuals with moderate to severe ID for variants in 565 known or candidate ID-associated genes using targeted next-generation sequencing. Likely pathogenic rare variants were found in ∼11% of the cases (113 variants in 107/986 individuals: ∼8% of the individuals had a likely pathogenic loss-of-function [LoF] variant, whereas ∼3% had a known pathogenic missense variant). Variants in SETD5, ATRX, CUL4B, MECP2, and ARID1B were the most common causes of ID. This study assessed the value of sequencing a cohort of probands to provide a molecular diagnosis of ID, without the availability of DNA from both parents for de novo sequence analysis. This modeling is clinically relevant as 28% of all UK families with dependent children are single parent households. In conclusion, to diagnose patients with ID in the absence of parental DNA, we recommend investigation of all LoF variants in known genes that cause ID and assessment of a limited list of proven pathogenic missense variants in these genes. This will provide 11% additional diagnostic yield beyond the 10%-15% yield from array CGH alone.Action Medical Research (SP4640); the Birth Defect Foundation (RG45448); the Cambridge National Institute for Health Research Biomedical Research Centre (RG64219); the NIHR Rare Diseases BioResource (RBAG163); Wellcome Trust award WT091310; The Cell lines and DNA bank of Rett Syndrome, X-linked mental retardation and other genetic diseases (member of the Telethon Network of Genetic Biobanks (project no. GTB12001); the Genetic Origins of Congenital Heart Disease Study (GO-CHD)- funded by British Heart Foundation (BHF)This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1002/humu.2290

MicroRNA biogenesis and their functions in regulating stem cell potency and differentiation

Stem cells are unspecialized/undifferentiated cells that exist in embryos and adult tissues or can be converted from somatic differentiated cells. Use of stem cells for tissue regeneration and tissue engineering has been a cornerstone of the regenerative medicine. Stem cells are also believed to exist in cancer tissues, namely cancer stem cells (CSCs). Growing evidence suggests that CSCs are the culprit of cancer dormancy, progression and recurrence, and thus have recently received great attention. MicroRNAs (miRNAs) are a group of short, non-coding RNAs that regulate expression of a wide range of genes at a post-transcriptional manner. They are emerging as key regulators of stem cell behaviors. This mini review summarizes the basic biogenesis and mode of actions of miRNAs, recent progress and discoveries of miRNAs in cellular reprogramming, stem cell differentiation and cellular communication, as well as miRNAs in CSCs. Some potential of miRNAs in future biomedical applications and research pertaining to stem cells are briefly discussed