Specific isoforms of translation initiation factor 4GI show differences in translational activity

The eukaryotic initiation factor (eIF) 4GI gene locus (eIF4GI) contains three identified promoters, generating alternately spliced mRNAs, yielding a total of five eIF4GI protein isoforms. Although eIF4GI plays a critical role in mRNA recruitment to the ribosomes, little is known about the functions of the different isoforms, their partner binding capacities, or the role of the homolog, eIF4GII, in translation initiation. To directly address this, we have used short interfering RNAs (siRNAs) expressed from DNA vectors to silence the expression of eIF4GI in HeLa cells. Here we show that reduced levels of specific mRNA and eIF4GI isoforms in HeLa cells promoted aberrant morphology and a partial inhibition of translation. The latter reflected dephosphorylation of 4E-BP1 and decreased eIF4F complex levels, with no change in eIF2 alpha phosphorylation. Expression of siRNA-resistant Myc-tagged eIF4GI isoforms has allowed us to show that the different isoforms exhibit significant differences in their ability to restore translation rates. Here we quantify the efficiency of eIF4GI promoter usage in mammalian cells and demonstrate that even though the longest isoform of eIF4GI (eIF4GIf) was relatively poorly expressed when reintroduced, it was more efficient at promoting the translation of cellular mRNAs than the more highly expressed shorter isoforms used in previous functional studies

Complexity and leadership in teacher professional development : the case of the National Centre for Excellence in the Teaching of Mathematics

There has been considerable interest in the teaching of mathematics over the last two decades, both internationally and in the UK. This has led to a number of government sponsored interventions in both curriculum and teacher professional development. The establishment of the National Centre for Excellence in the Teaching of Mathematics (NCETM) in 2006, arguably, represented a departure from previous policy and initiatives related to professional development for teachers of mathematics. This paper looks at what was distinctive about the NCETM approach (2006-2010) and the impact of its work, as well as exploring a number of theoretical issues that arise when describing these. The paper draws on data from a study of the impact of the NCETM that was informed by interview and case studies. Telephone interviews were conducted with 89 teachers and others with differing levels of involvement with the NCETM. In addition, 10 school-based case studies of different NCETM-supported activity were conducted. This material was analysed using a CPD evaluation model (Coldwell and Simkins, 2011) and more generally in relation to literature on school and teacher change. In this paper, we explore ways in which theoretical tools drawn from complexity theory - complex adaptive systems and formal and informal systems - can be used to describe the nature and consequences of the NCETM's actions. Further, in understanding and assessing the impact of the NCETM intervention on subject leadership and teacher identity we suggest that parallels can be made with analyses of identity in social movements. Finally, we examine the concepts of dispersed and distributed leadership in relation to their applicability to the organic development of mathematics teacher leadership that the NCETM promotes. The paper outlines both the type of outcomes of the NCETM's activity and the factors that supported these. Many of these are similar to those previously identified in relation to professional development that focuses on and supports school-based leadership and can be analysed in terms of theoretical concepts such as distributed and dispersed leadership. However, the NCETM's approach had some distinctive impacts and features that, we contend, are particular to the complex interrelationship of the different forms of NCETM activity

Rethinking models of professional learning as tools: a conceptual analysis to inform research and practice

One approach to designing, researching or evaluating professional learning experiences is to use models of learning processes. Here we analyse and critique five significant contemporary analytical models: three variations on path models, proposed by Guskey, Desimone and Clarke and Hollingsworth; a model using a systemic conceptualisation of learning by Opfer and Pedder; and a cognitive learning model by Evans. To do this, we develop and illustrate an analytical framework focused on model components, purposes, scope, explicit and implicit theories of learning and change processes, agency and philosophical underpinnings. We identify similarities, differences, inconsistencies and limitations in the models. This provides the basis for reconceptualising models as tools to be deployed alongside other relevant constructs and thus the analytical framework can support a more informed selection of theoretical models by researchers and practitioners

Connecting research and teacher education : quality enhancement for ITE Partnerships

As part of the Welsh education reform programme, the relationship between research and teacher education has been identified as an important component for improving Welsh education and meeting the aspirations of the new Welsh Curriculum. The new ITE accreditation criteria (Welsh Government, 2017) seek to contribute to meeting the challenge to move towards matching internationally excellent practice in research-rich teacher education in a form appropriate to Welsh contexts. Quality enhancement is a change process in which ongoing evaluation is embedded and used to inform development. It is informed by both theory based (Weiss, 1997; Rogers, 2008) and realistic evaluation methodologies (Pawson & Tilley, 1997) adapted for improvement planning. The quality enhancement tool presented here is designed to support ITE partnerships to meet the ITE accreditation criteria and engage in ongoing quality enhancement. It will support providers and potential providers to set aims and goals, articulate change mechanisms and identify resources needed and actions to take, use both external and institutional evidence to review the current situation, plan the monitoring of progress. The intended users of the tool are those responsible for planning and leading ITE programmes in ITE partnerships. The tool will be used most effectively through a process of dialogue and collaboration with all stakeholders within institutions and with school and other external partners. This collaboration with schools is essential in relation to areas where responsibility is shared for the quality of ITE

Phosphorylation of eIF4GII and 4E-BP1 in response to nocodazole treatment: a reappraisal of translation initiation during mitosis

Translation mechanisms at different stages of the cell cycle have been studied for many years, resulting in the dogma that translation rates are slowed during mitosis, with cap-independent translation mechanisms favored to give expression of key regulatory proteins. However, such cell culture studies involve synchronization using harsh methods, which may in themselves stress cells and affect protein synthesis rates. One such commonly used chemical is the microtubule de-polymerization agent, nocodazole, which arrests cells in mitosis and has been used to demonstrate that translation rates are strongly reduced (down to 30% of that of asynchronous cells). Using synchronized HeLa cells released from a double thymidine block (G 1/S boundary) or the Cdk1 inhibitor, RO3306 (G 2/M boundary), we have systematically re-addressed this dogma. Using FACS analysis and pulse labeling of proteins with labeled methionine, we now show that translation rates do not slow as cells enter mitosis. This study is complemented by studies employing confocal microscopy, which show enrichment of translation initiation factors at the microtubule organizing centers, mitotic spindle, and midbody structure during the final steps of cytokinesis, suggesting that translation is maintained during mitosis. Furthermore, we show that inhibition of translation in response to extended times of exposure to nocodazole reflects increased eIF2α phosphorylation, disaggregation of polysomes, and hyperphosphorylation of selected initiation factors, including novel Cdk1-dependent N-terminal phosphorylation of eIF4GII. Our work suggests that effects on translation in nocodazole-arrested cells might be related to those of the treatment used to synchronize cells rather than cell cycle status

An Evaluation of the Further Mathematics Support Programme : Research Report

The four key focus areas for the 2014-16 evaluation were to consider: • capacity and capability building • reach to schools/colleges, teachers and students • effectiveness of the programme (quality and impact) • sustainability beyond the end of the programme The evaluation also sought to assess the viability and value of quantitative modelling of Further Mathematics and FMSP activity, including identifying any relevant issues in drawing together disparate databases, to provide a quantitative baseline for future evaluation and to develop tools for future evaluation, including assessing the value of focus group interviews with students as a data collection method. The evaluation affirms previous findings about the quality and value of FMSP activity and the positive regard of stakeholders for the programme. It supports a continuation of a varied programme offer. The analysis highlights the important role that the FMSP has and can play in supporting Further Mathematics culture particularly in contexts and centres in challenging circumstances. The evaluation confirms the impact that the FMSP has had on widening participation in Further Mathematics, but also indicates that access to Further Mathematics - and so to both the intrinsic benefits of this as well as access to further opportunities - continues to be more available to students who are socially and economically advantaged

The human insulin receptor mRNA contains a functional internal ribosome entry segment.

Regulation of mRNA translation is an important mechanism determining the level of expression of proteins in eukaryotic cells. Translation is most commonly initiated by cap-dependent scanning, but many eukaryotic mRNAs contain internal ribosome entry segments (IRESs), providing an alternative means of initiation capable of independent regulation. Here, we show by using dicistronic luciferase reporter vectors that the 5'-UTR of the mRNA encoding human insulin receptor (hIR) contains a functional IRES. RNAi-mediated knockdown showed that the protein PTB was required for maximum IRES activity. Electrophoretic mobility shift assays confirmed that PTB1, PTB2 and nPTB, but not unr or PTB4, bound to hIR mRNA, and deletion mapping implicated a CCU motif 448 nt upstream of the initiator AUG in PTB binding. The IR-IRES was functional in a number of cell lines, and most active in cells of neuronal origin, as assessed by luciferase reporter assays. The IRES was more active in confluent than sub-confluent cells, but activity did not change during differentiation of 3T3-L1 fibroblasts to adipocytes. IRES activity was stimulated by insulin in sub-confluent cells. The IRES may function to maintain expression of IR protein in tissues such as the brain where mRNA translation by cap-dependent scanning is less effective

SCOPE New Photographic Practices

The photographic practices brought together for this exhibition and publication provide a broad scope of how photographic and lens based media may be used in order to have a visceral and conceptual impact. The methods on show demonstrate the way that artists might pick and choose from the approaches, processes and debates that have arisen through the medium’s history. This collection of work features film, video and photography that demand a renegotiation of the relationship between camera, subject and viewer. Visual Art Centre Gallery, Tsinghua University, Beijing, Chin

PEITC-mediated inhibition of mRNA translation is associated with both inhibition of mTORC1 and increased eIF2α phosphorylation in established cell lines and primary human leukemia cells.

Increased mRNA translation drives carcinogenesis and is an attractive target for the development of new anti-cancer drugs. In this work, we investigated effects of phenethylisothiocyanate (PEITC), a phytochemical with chemopreventive and anti-cancer activity, on mRNA translation. PEITC rapidly inhibited global mRNA translation in human breast cancer-derived MCF7 cells and mouse embryonic fibroblasts (MEFs). In addition to the known inhibitory effects of PEITC on mTORC1 activity, we demonstrate that PEITC increased eIF2α phosphorylation. PEITC also increased formation of stress granules which are typically associated with eIF2α phosphorylation and accumulation of translationally stalled mRNAs. Analysis of genetically modified MEFs demonstrated that optimal inhibition of global mRNA translation by PEITC was dependent on eIF2α phosphorylation, but not mTORC1 inhibition. We extended this study into primary leukemic B cells derived from patients with chronic lymphocytic leukaemia (CLL). CLL cells were stimulated in vitro with anti-IgM to mimic binding of antigen, a major driver of this leukemia. In CLL cells, PEITC increased eIF2α phosphorylation, inhibited anti-IgM-induced mTORC1 activation and decreased both basal and anti-IgM-induced global mRNA translation. PEITC also inhibited transcription and translation of MYC mRNA and accumulation of the MYC oncoprotein, in anti-IgM-stimulated cells. Moreover, treatment of CLL cells with PEITC and the BTK kinase inhibitor ibrutinib decreased anti-IgM-induced translation and induced cell death to a greater extent than either agent alone. Therefore, PEITC can inhibit both global and mRNA specific translation (including MYC) via effects on multiple regulatory pathways. Inhibition of mRNA translation may contribute to the chemopreventive and anti-cancer effects of PEITC

Compartmentalisation and localisation of the translation initiation factor (eIF) 4F complex in normally growing fibroblasts

Previous observations of association of mRNAs and ribosomes with subcellular structures highlight the importance of localised translation. However, little is known regarding associations between eukaryotic translation initiation factors and cellular structures within the cytoplasm of normally growing cells. We have used detergent-based cellular fractionation coupled with immunofluorescence microscopy to investigate the subcellular localisation in NIH3T3 fibroblasts of the initiation factors involved in recruitment of mRNA for translation, focussing on eIF4E, the mRNA cap-binding protein, the scaffold protein eIF4GI and poly(A) binding protein (PABP). We find that these proteins exist mainly in a soluble cytosolic pool, with only a subfraction tightly associated with cellular structures. However, this "associated" fraction was enriched in active "eIF4F" complexes (eIF4E.eIF4G.eIF4A.PABP). Immunofluorescence analysis reveals both a diffuse and a perinuclear distribution of eIF4G, with the perinuclear staining pattern similar to that of the endoplasmic reticulum. eIF4E also shows both a diffuse staining pattern and a tighter perinuclear stain, partly coincident with vimentin intermediate filaments. All three proteins localise to the lamellipodia of migrating cells in close proximity to ribosomes, microtubules, microfilaments and focal adhesions, with eIF4G and eIF4E at the periphery showing a similar staining pattern to the focal adhesion protein vinculin