Yeast m6 A 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-methyladenosine (m6 A) deaminase activity, thus suggesting a link between obesity-associated diseases and the presence of m6 A 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 m6 A 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 m6 A 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 m6 A and message translatability. We also find m6 A induction following prolonged rapamycin treatment

eIF4A Inhibition Allows Translational Regulation of mRNAs Encoding Proteins Involved in Alzheimer's Disease

Alzheimer's 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 Alzheimer's disease: reducing A-beta and tau synthesis, while increasing proteins predicted to be neuroprotective

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

Homoeologous gene silencing in tissue cultured wheat callus

Abstract Background In 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. Results Evidence 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. Conclusion Our 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.</p

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

Patterns of gene silencing in hexaploid wheat

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Overlaps between the yeast methylome and the polysome fractions.

<p>809 overlapping genes were identified between the yeast methylome dataset(1181 genes; all duplications were removed) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132090#pone.0132090.ref017" target="_blank">17</a>] and the genelist for ribosome association (4094 significant genes) used for cluster analysis. The gene list overlaps were determined using the BioVenn web based tool].</p

Cluster analysis of transcript polysome occupancy.

<p>Total RNA from the different pooled sucrose gradient fractions were extracted and prepared for Affymetrix analysis. The input sample is the non-fractionated total RNA sample. The data for vegetative samples were ‘.cell’ files from Dominissini et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132090#pone.0132090.ref006" target="_blank">6</a>] The raw chip data were analysed using Partek GS 6.0 software package. The gene list of significant genes (FDR<0.05, no fold change cut off was applied) was the base for hierarchic clustering using Partek GS 6.0. 12 different clusters were identified (labelled by different colour bars) (a). The gene lists for each different cluster were used to determine the enrichment for methylated transcripts using the yeast methylome from Schwartz <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132090#pone.0132090.ref017" target="_blank">17</a>]. The statistical significance of the overlaps was determined using the online resource <a href="http://nemates.org/MA/progs/overlap_stats.html" target="_blank">http://nemates.org/MA/progs/overlap_stats.html</a> (b).</p