Andrew Bottley

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.

Yeast m6A Methylated mRNAs Are Enriched on Translating Ribosomes during Meiosis, and under Rapamycin Treatment

Interest in mRNA methylation has exploded in recent years. The sudden interest in a 40 year old discovery was due in part to the finding of FTO’s (Fat Mass Obesity) N6-methyl-adenosine (m6A) deaminase activity, thus suggesting a link between obesity-associated diseases and the presence of m6A in mRNA. Another catalyst of the sudden rise in mRNA methylation research was the release of mRNA methylomes for human, mouse and Saccharomyces cerevisiae. However, the molecular function, or functions of this mRNA ‘epimark’ remain to be discovered. There is supportive evidence that m6A could be a mark for mRNA degradation due to its binding to YTH domain proteins, and consequently being chaperoned to P bodies. Nonetheless, only a subpopulation of the methylome was found binding to YTHDF2 in HeLa cells.The model organism Saccharomyces cerevisiae, has only one YTH domain protein (Pho92, Mrb1), which targets PHO4 transcripts for degradation under phosphate starvation. However, mRNA methylation is only found under meiosis inducing conditions, and PHO4 transcripts are apparently non-methylated. In this paper we set out to investigate if m6A could function alternatively to being a degradation mark in S. cerevisiae; we also sought to test whether it can be induced under non-standard sporulation conditions. We find a positive association between the presence of m6A and message translatability. We also find m6A induction following prolonged rapamycin treatment.

Transcript profiling of the hypomethylated hog1 mutant of Arabidopsis

Transcript profiling was used to look for genes that differ in expression between the SAH hydrolase deficient and hypomethylated hog1-1 mutant and the parental (HOG1) line. This analysis identified a subset of gene transcripts that were up-regulated in hog1-1 plants. The majority of these transcripts were from genes located in the pericentromeric heterochromatin. About a third of the genes are annotated as transposons or having transposon homology. Subsequent experiments using Northern blots, RT-PCR and real-time RT-PCR confirmed the up-regulation of 19 of the genes and identified a set of molecular probes for genes that are up-regulated in the hog1-1 background. Six (of six genes tested) of the hog1-1 up-regulated genes are also up-regulated in the hypomethylated ddm1 mutant, three in the hypomethylated met1 mutant and three in the dcl3 mutant. The results suggest that the hypomethylation in the mutant lines may have a causal role in the up-regulation of these transcripts.

eIF4A inhibition allows translational regulation of mRNAs encoding proteins involved in Alzheimer's disease.

Alzheimers disease (AD) is the main cause of dementia in our increasingly aging population. The debilitating cognitive and behavioral symptoms characteristic of AD make it an extremely distressing illness for patients and carers. Although drugs have been developed to treat AD symptoms and to slow disease progression, there is currently no cure. The incidence of AD is predicted to increase to over one hundred million by 2050, placing a heavy burden on communities and economies, and making the development of effective therapies an urgent priority. Two proteins are thought to have major contributory roles in AD: the microtubule associated protein tau, also known as MAPT; and the amyloid-beta peptide (A-beta), a cleavage product of amyloid precursor protein (APP). Oxidative stress is also implicated in AD pathology from an early stage. By targeting eIF4A, an RNA helicase involved in translation initiation, the synthesis of APP and tau, but not neuroprotective proteins, can be simultaneously and specifically reduced, representing a novel avenue for AD intervention. We also show that protection from oxidative stress is increased upon eIF4A inhibition. We demonstrate that the reduction of these proteins is not due to changes in mRNA levels or increased protein degradation, but is a consequence of translational repression conferred by inhibition of the helicase activity of eIF4A. Inhibition of eIF4A selectively and simultaneously modulates the synthesis of proteins involved in Alzheimers disease: reducing A-beta and tau synthesis, while increasing proteins predicted to be neuroprotective.

PRAME Is a Golgi-Targeted Protein That Associates with the Elongin BC Complex and Is Upregulated by Interferon-Gamma and Bacterial PAMPs

Preferentially expressed antigen in melanoma (PRAME) has been described as a cancer-testis antigen and is associated with leukaemias and solid tumours. Here we show that PRAME gene transcription in leukaemic cell lines is rapidly induced by exposure of cells to bacterial PAMPs (pathogen associated molecular patterns) in combination with type 2 interferon (IFNγ). Treatment of HL60 cells with lipopolysaccharide or peptidoglycan in combination with IFNγ resulted in a rapid and transient induction of PRAME transcription, and increased association of PRAME transcripts with polysomes. Moreover, treatment with PAMPs/IFNγ also modulated the subcellular localisation of PRAME proteins in HL60 and U937 cells, resulting in targeting of cytoplasmic PRAME to the Golgi. Affinity purification studies revealed that PRAME associates with Elongin B and Elongin C, components of Cullin E3 ubiquitin ligase complexes. This occurs via direct interaction of PRAME with Elongin C, and PRAME colocalises with Elongins in the Golgi after PAMP/IFNγ treatment. PRAME was also found to co-immunoprecipitate core histones, consistent with its partial localisation to the nucleus, and was found to bind directly to histone H3 in vitro. Thus, PRAME is upregulated by signalling pathways that are activated in response to infection/inflammation, and its product may have dual functions as a histone-binding protein, and in directing ubiquitylation of target proteins for processing in the Golgi.

Homoeologous gene silencing in tissue cultured wheat callus

BackgroundIn contrast to diploids, most polyploid plant species, which include the hexaploid bread wheat, possess an additional layer of epigenetic complexity. Several studies have demonstrated that polyploids are affected by homoeologous gene silencing, a process in which sub-genomic genomic copies are selectively transcriptionally inactivated. This form of silencing can be tissue specific and may be linked to developmental or stress responses.ResultsEvidence was sought as to whether the frequency of homoeologous silencing in in vitro cultured wheat callus differ from that in differentiated organs, given that disorganized cells are associated with a globally lower level of DNA methylation. Using a reverse transcription PCR (RT-PCR) single strand conformation polymorphism (SSCP) platform to detect the pattern of expression of 20 homoeologous sets of single-copy genes known to be affected by this form of silencing in the root and/or leaf, we observed no silencing in any of the wheat callus tissue tested.ConclusionOur results suggest that much of the homoeologous silencing observed in differentiated tissues is probably under epigenetic control, rather than being linked to genomic instability arising from allopolyploidization. This study reinforces the notion of plasticity in the wheat epi-genome.

Epigenetic Variation Amongst Polyploidy Crop Species

Many agronomically important crop species such as wheat are (or were once) polyploid, with at least one round of whole genome duplication occurring before domestication. This genetic buffering or redundancy allows for sequence divergence, and in turn the development of functional variations between duplicated genes (homoeologues). Homoeologues may encode proteins with different properties and plant breeders have successfully used this genetic resource to introduce new genetic diversity into breeding populations. However duplicated genes are also subject to extensive epigenetic control and are therefore not always equally expressed. The preferential bias in the expression or the silencing of a specific homoeologue may be heritable and can be stable across many generations. There is also mounting evidence to suggest that selective homoeologue expression occurs in response to stresses such as salinity and may be specific to individual pathways or processes. Importantly, this type of epigenetic variation may segregate within a breeding population and is readily observed in newly synthesised polyploid hybrids.

Methylation profiling RIN3 and MEF2C identifies epigenetic marks associated with sporadic early onset Alzheimer’s disease

A number of genetic loci associate with early onset Alzheimer’s disease (EOAD); however, the drivers of this disease remains enigmatic. Genome wide association and in vivo modeling have shown that loss-of-function, e.g., ABCA7, reduced levels of SIRT1 and MEFF2C, or increased levels of PTK2β confer risk or link to the pathogenies. It is known that DNA methylation can profoundly affect gene expression and can impact on the composition of the proteome; therefore, the aim of this study is to assess if genes associated with sporadic EOAD (sEOAD) are differentially methylated. Epi-profiles of DNA extracted from blood and cortex were compared using a pyrosequencing platform. We identified significant group-wide hypomethylation in AD blood when compared to controls for 7 CpGs located within the 3’UTR of RIN3 (CpG1 p = 0.019, CpG2 p = 0.018, CpG3 p = 0.012, CpG4 p = 0.009, CpG5 p = 0.002, CpG6 p = 0.018, and CpG7 p = 0.013, respectively; AD/Control n = 22/26; Male/Female n = 27/21). Observed effects were not gender specific. No group wide significant differences were found in the promoter methylation of PTK2β, ABCA7, SIRT1, or MEF2C, genes known to associate with late onset AD. A rare and significant difference in methylation was observed for one CpG located upstream of the MEF2C promoter in one AD individual only (22% reduction in methylation, p = 2.0E-10; Control n = 26, AD n = 25, Male/Female n = 29/22). It is plausible aberrant methylation may mark sEOAD in blood and may manifest in some individuals as rare epi-variants for genes linked to sEOAD.

Aberrant translation of proteins implicated in Alzheimer's disease pathology

Introduction Control of protein manufacture at the point of translation is a crucial step in the regulation of gene expression and has shown to be important to many neurological processes, for example, synaptic plasticity and memory formation. The aim of this review was to discuss aberrant translation of proteins implicated in Alzheimer’s disease pathology. Discussion Aberrant translation has been linked with neurodegenerative conditions such as Alzheimer’s disease; however, it is not fully understood how this aberrant protein synthesis occurs or how this may be sustained in Alzheimer’s disease. Cell stressors such as oxidative stress may enhance the translation of Alzheimer’s disease– associated proteins (e.g. amyloid precursor protein), and new research suggests that the cell survival response (e.g. elF2-alpha phosphorylation) may inadvertently up-regulate the translation of proteins such as BACE1; a process mediated in this instance by an upstream open reading frame located within the BACE1 5’UTR. Conclusion The research discussed in this review article has identified that in addition to regulation at the point of transcription and post-translational protein processing, the levels of proteins which negatively associate with Alzheimer’s disease pathology may also be controlled at the point of translation. Stressors such as oxidative stress may drive the transcription of amyloid precursor protein and the cleavage of amyloid precursor protein and may also enhance the translational efficiency of both amyloid precursor protein and the secretase responsible for cleaving amyloid precursor protein into its cytotoxic Abeta42 fragment. We suggest that selectively inhibiting the translation machinery in combination with reducing the levels of oxidative stress may represent a new therapeutic avenue for the treatment of Alzheimer’s disease. Introduction Translation Gene expression comprises a series of complex and tightly regulated events, starting with transcription and ending with protein synthesis. In a simple model, translation initiation of messenger RNA begins when the cap-binding protein complex is recruited to the cap complex. This complex then scans the 5’UTR of the messenger RNA in a 5ʹ to 3ʹ direction until a start codon is recognised and synthesis is initiated (reviewed in depth by Jackson et al.1); the process of translation then progresses through three stages: initiation, elongation, and termination. The cap-binding complex that forms at the initiation step is an assembly of a multitude of translation initiation factors, while the 5’-untranslated region (5’UTR) may differ in length, possess features such as tertiary structures, upstream open reading frames (uORFs) and RNA proteinbinding sites2–4. Importantly, each feature may act as a regulatory element independently enhancing or repressing the rate of protein synthesis (see Jackson et al.1). Transcription and mRNA processing can be a lengthy process; therefore regulation of gene expression at the point of transcription may be insufficiently rapid to function as a responder to a stimuli or stress. One hypothesis suggests that the cell can overcome this constraint by transcribing reservoirs of inactive messages that remain dormant awaiting translational activation only when protein is required—an idea supported by data showing that mRNA levels only partly correlate with protein levels5. This model allows for transcripts to be ‘activated’, then proteins rapidly synthesised from stored templates. It is unsurprising that most detailed investigations into the regulation of translation have focussed on the area of stress responses, where the rate of protein manufacture may directly determine cell survival. However, recent research has also identified this step in the gene expression pathway to be a key stage of control in the manufacture of proteins linked with the pathology of progressive neurodegenerative conditions such as Alzheimer’s disease (AD). The aim of this review was to discuss new and exciting research that identifies putative mechanisms of regulation of proteins associated with AD. Discussion The authors have referenced some of their own studies in this review. The protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed. * Corresponding author Email: Andrew.bottley@nottingham.ac.uk; Alexander.kondrashov@nottingham.ac.uk 1 School of Biosciences, University of Nottingham, Nottingham, UK 2 School of Medicine, University of Nottingham, Nottingham, UK