ifet-1 is a broad-scale translational repressor required for normal P granule formation in C. elegans

Large cytoplasmic ribonucleoprotein germ granule complexes are a common feature in germ cells. In C. elegans these are called P granules and for much of the life-cycle they associate with nuclear pore complexes in germ cells. P granules are rich in proteins that function in diverse RNA pathways. Here we report that the C. elegans homolog of the eIF4E-transporter IFET-1 is required for oogenesis but not spermatogenesis. We show that IFET-1 is required for translational repression of several maternal mRNAs in the distal gonad and functions in conjunction with the broad-scale translational regulators CGH-1, CAR-1 and PATR-1 to regulate germ cell sex determination. Furthermore we have found that IFET-1 localizes to P granules throughout the gonad and in the germ cell lineage in the embryo. Interestingly, IFET-1 is required for the normal ultrastructure of P granules and for the localization of CGH-1 and CAR-1 to P granules. Our findings suggest that IFET-1 is a key translational regulator and is required for normal P granule formation.Madhu S. Sengupta, Wai Yee Low, Joseph R. Patterson, Hyun-Min Kim, Ana Traven, Traude H. Beilharz, Monica P. Colaia, covo, Jennifer A. Schisa, and Peter R. Boa

microRNA-Mediated Messenger RNA Deadenylation Contributes to Translational Repression in Mammalian Cells

Animal microRNAs (miRNAs) typically regulate gene expression by binding to partially complementary target sites in the 3′ untranslated region (UTR) of messenger RNA (mRNA) reducing its translation and stability. They also commonly induce shortening of the mRNA 3′ poly(A) tail, which contributes to their mRNA decay promoting function. The relationship between miRNA-mediated deadenylation and translational repression has been less clear. Using transfection of reporter constructs carrying three imperfectly matching let-7 target sites in the 3′ UTR into mammalian cells we observe rapid target mRNA deadenylation that precedes measureable translational repression by endogenous let-7 miRNA. Depleting cells of the argonaute co-factors RCK or TNRC6A can impair let-7-mediated repression despite ongoing mRNA deadenylation, indicating that deadenylation alone is not sufficient to effect full repression. Nevertheless, the magnitude of translational repression by let-7 is diminished when the target reporter lacks a poly(A) tail. Employing an antisense strategy to block deadenylation of target mRNA with poly(A) tail also partially impairs translational repression. On the one hand, these experiments confirm that tail removal by deadenylation is not strictly required for translational repression. On the other hand they show directly that deadenylation can augment miRNA-mediated translational repression in mammalian cells beyond stimulating mRNA decay. Taken together with published work, these results suggest a dual role of deadenylation in miRNA function: it contributes to translational repression as well as mRNA decay and is thus critically involved in establishing the quantitatively appropriate physiological response to miRNAs

microRNA input into a neural ultradian oscillator controls emergence and timing of alternative cell states.

© 2014 Macmillan Publishers LimitedThis is an open access article that is freely available in ORE or from the publisher's web site. Please cite the published version.Progenitor maintenance, timed differentiation and the potential to enter quiescence are three fundamental processes that underlie the development of any organ system. In the nervous system, progenitor cells show short-period oscillations in the expression of the transcriptional repressor Hes1, while neurons and quiescent progenitors show stable low and high levels of Hes1, respectively. Here we use experimental data to develop a mathematical model of the double-negative interaction between Hes1 and a microRNA, miR-9, with the aim of understanding how cells transition from one state to another. We show that the input of miR-9 into the Hes1 oscillator tunes its oscillatory dynamics, and endows the system with bistability and the ability to measure time to differentiation. Our results suggest that a relatively simple and widespread network of cross-repressive interactions provides a unifying framework for progenitor maintenance, the timing of differentiation and the emergence of alternative cell states.Wellcome Trus

Partial Depletion of Natural CD4+CD25+ Regulatory T Cells with Anti-CD25 Antibody Does Not Alter the Course of Acute Influenza A Virus Infection

Foxp3+ CD4+ regulatory T cells represent a T cell subset with well-characterized immunosuppressive effects during immune homeostasis and chronic infections, and there is emerging evidence to suggest these cells temper pulmonary inflammation in response to acute viral infection. Recent studies have demonstrated treatment with PC61 CD25-depleting antibody potentiates inflammation in a murine model of RSV infection, while paradoxically delaying recruitment of CD8+ T cells to the site of inflammation. The present study therefore sought to examine the role of these cells in a murine model of acute influenza A virus infection through the administration of PC61 CD25-depleting antibody. PC61 antibody is able to partially deplete CD25+Foxp3+ regulatory T cells to a comparable degree as seen within previous work examining RSV, however this does not alter influenza A-virus induced mortality, weight loss, viral clearance and cellularity within the lung. Collectively, these data demonstrate that partial depletion of CD4+CD25+ regulatory T cells with PC61 antibody does not alter the course of influenza A virus infection

The Unseeing Eye: Disability and the hauntology of Derrida’s ghost. A story in three parts

Through the employment of the three stanzas of Thomas Hardy’s poem ‘The Self-Unseeing’ this paper seeks to tremble the picture of disability located in the pedagogical materials in English Schools. By mobilising, and then reversing, Derrida’s concept of the visor and the ghost, as well as Bentham’s Panopticon, this story reveals the power of the Them, the Their and the They. In materialising the ghost of the real of disability within a utopia of hope this story deconstructs the power of Their transparent house by revealing disabled people as magnificent beings

Post-transcriptional gene regulation: From genome-wide studies to principles

Post-transcriptional regulation of gene expression plays important roles in diverse cellular processes such as development, metabolism and cancer progression. Whereas many classical studies explored the mechanistics and physiological impact on specific mRNA substrates, the recent development of genome-wide analysis tools enables the study of post-transcriptional gene regulation on a global scale. Importantly, these studies revealed distinct programs of RNA regulation, suggesting a complex and versatile post-transcriptional regulatory network. This network is controlled by specific RNA-binding proteins and/or non-coding RNAs, which bind to specific sequence or structural elements in the RNAs and thereby regulate subsets of mRNAs that partly encode functionally related proteins. It will be a future challenge to link the spectra of targets for RNA-binding proteins to post-transcriptional regulatory programs and to reveal its physiological implications

Seamless Gene Tagging by Endonuclease-Driven Homologous Recombination

Gene tagging facilitates systematic genomic and proteomic analyses but chromosomal tagging typically disrupts gene regulatory sequences. Here we describe a seamless gene tagging approach that preserves endogenous gene regulation and is potentially applicable in any species with efficient DNA double-strand break repair by homologous recombination. We implement seamless tagging in Saccharomyces cerevisiae and demonstrate its application for protein tagging while preserving simultaneously upstream and downstream gene regulatory elements. Seamless tagging is compatible with high-throughput strain construction using synthetic genetic arrays (SGA), enables functional analysis of transcription antisense to open reading frames and should facilitate systematic and minimally-invasive analysis of gene functions

Deregulation of MYCN, LIN28B and LET7 in a Molecular Subtype of Aggressive High-Grade Serous Ovarian Cancers

Molecular subtypes of serous ovarian cancer have been recently described. Using data from independent datasets including over 900 primary tumour samples, we show that deregulation of the Let-7 pathway is specifically associated with the C5 molecular subtype of serous ovarian cancer. DNA copy number and gene expression of HMGA2, alleles of Let-7, LIN28, LIN28B, MYC, MYCN, DICER1, and RNASEN were measured using microarray and quantitative reverse transcriptase PCR. Immunohistochemistry was performed on 127 samples using tissue microarrays and anti-HMGA2 antibodies. Fluorescence in situ hybridisation of bacterial artificial chromosomes hybridized to 239 ovarian tumours was used to measure translocation at the LIN28B locus. Short interfering RNA knockdown in ovarian cell lines was used to test the functionality of associations observed. Four molecular subtypes (C1, C2, C4, C5) of high-grade serous ovarian cancers were robustly represented in each dataset and showed similar pattern of patient survival. We found highly specific activation of a pathway involving MYCN, LIN28B, Let-7 and HMGA2 in the C5 molecular subtype defined by MYCN amplification and over-expression, over-expression of MYCN targets including the Let-7 repressor LIN28B, loss of Let-7 expression and HMGA2 amplification and over-expression. DICER1, a known Let-7 target, and RNASEN were over-expressed in C5 tumours. We saw no evidence of translocation at the LIN28B locus in C5 tumours. The reported interaction between LIN28B and Let-7 was recapitulated by siRNA knockdown in ovarian cancer cell lines. Our results associate deregulation of MYCN and downstream targets, including Let-7 and oncofetal genes, with serous ovarian cancer. We define for the first time how elements of an oncogenic pathway, involving multiple genes that contribute to stem cell renewal, is specifically altered in a molecular subtype of serous ovarian cancer. By defining the drivers of a molecular subtype of serous ovarian cancers we provide a novel strategy for targeted therapeutic intervention