Dcp1-Bodies in Mouse Oocytes

Processing bodies (P-bodies) are cytoplasmic granules involved in the storage and degradation of mRNAs. In somatic cells, their formation involves miRNA-mediated mRNA silencing. Many P-body protein components are also found in germ cell granules, such as in mammalian spermatocytes. In fully grown mammalian oocytes, where changes in gene expression depend entirely on translational control, RNA granules have not as yet been characterized. Here we show the presence of P-body-like foci in mouse oocytes, as revealed by the presence of Dcp1a and the colocalization of RNA-associated protein 55 (RAP55) and the DEAD box RNA helicase Rck/p54, two proteins associated with P-bodies and translational control. These P-body-like structures have been called Dcp1-bodies and in meiotically arrested primary oocytes, two types can be distinguished based on their size. They also have different protein partners and sensitivities to the depletion of endogenous siRNA/miRNA and translational inhibitors. However, both type progressively disappear during in vitro meiotic maturation and are virtually absent in metaphase II–arrested secondary oocytes. Moreover, this disassembly of hDcp1a-bodies is concomitant with the posttranslational modification of EGFP-hDcp1a

An AUG Codon Conserved for Protein Function Rather than Translational Initiation: The Story of the Protein sElk1

<div><p>Elk1 belongs to the ternary complex (TCF) subfamily of the ETS-domain transcription factors. Several studies have implicated an important function for Elk1 in the CNS including synaptic plasticity and cell differentiation. Whilst studying <i>ELK1</i> gene expression in rat brain a 54 aa N-terminally truncated isoform lacking the DBD was observed on immunoblots. A similar protein was also detected in NGF differentiated PC12 cells. It was proposed that this protein, referred to as sElk1, arose due to a <i>de-novo</i> initiation event at the second AUG codon on the Elk1 ORF. Transient over-expression of sElk1 potentiated neurite growth in the PC12 model and induced differentiation in the absence of NGF, leading to the proposition that it may have a specific function in the CNS. Here we report on the translational expression from the mouse and rat transcript and compare it with our earlier published work on human. Results demonstrate that the previously observed sElk1 protein is a non-specific band arising from the antibody employed. The tight conservation of the internal AUG reported to drive sElk1 expression is in fact coupled to Elk1 protein function, a result consistent with the Elk1-SRE crystal structure. It is also supported by the observed conservation of this methionine in the DBD of all ETS transcription factors independent of the N- or C-terminal positioning of this domain. Reporter assays demonstrate that elements both within the 5′UTR and downstream of the AUG^Elk1 serve to limit 40S access to the AUG^sElk1 codon.</p></div

Function of the methionine 55.

<p>(A). Organisation of the Elk1 protein. Upper panel: Protein functional domains (ETS  =  DNA binding, B =  SRF interaction, D =  MAPK docking site, TAD  =  transactivation domain). Middle panel: Alignment of the Elk1 ETS domain from mouse, rat and human. The residues that make conserved DNA backbone and base contacts and are common to both Elk1 and SAP1 are indicated by the inverted triangles. The methionine corresponding to the AUG^sElk1 is boxed in grey <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102890#pone.0102890-Mo1" target="_blank">[10]</a>. Lower panel: A consensus ETS domain based upon the alignment of 12 family members with DBDs both N- and C-terminal. Tightly conserved amino acids are indicated in bold capitol. The methionine corresponding to the AUG^sELK1 is boxed in grey (adapted from <a href="http://content.lib.utah.edu/utils/getfile/collection/etd1/id/1528/filename/1687.pdf" target="_blank">http://content.lib.utah.edu/utils/getfile/collection/etd1/id/1528/filename/1687.pdf</a>) (B). Chip analysis at the c-fos promoter. HEK293T cells were transfected with plasmids expressing ELK1^HA, sElk1^HA or the Elk1^HA M55S mutant. ChIP analysis coupled to qPCR was performed as outlined in the supplemental section. The assay was performed twice (the duplicate columns) and analysed by qPCR. The ΔCt values (relative to input) were then normalised relative to Elk1^HA which was given a value of 1 in each experiment. (C). Immunoblot confirming expression from the transfected plasmids. Mock indicates non-transfected cells.</p

Characterisation of the <i>ELK1</i> gene.

<p>(A). Alignment of the genomic sequences at the 5′ extremity of the <i>ELK1</i> gene between Human (Hsap), Mouse (Mmus) and Rat (Rnor). Conserved bases are highlighted in grey. The sequence referred to as exons in the Ensembl Database are in red and the sequence of the exon II/III that we cloned and sequenced from PC12 cells is presented in green. The positions of the uAUG1, uAUG2 (boxed in blue) and AUG^Elk1 are indicated. The * indicates the splice boundaries of the rat exon II. (B). Upper portion: Exonic organisation of the human and rat <i>ELK1</i> gene with the sizes of each exon (bps) and the positioning of key AUG codons indicated. Lower portion: A schematic representation that highlight's the conservation in the organisation of the 5′UTR^L and 5′UTR^S<i>ELK1</i> transcripts in both human and rat. SL1 and SL2 refer to the stable stem-loop structures around uAUG1 and 14 nts is the spacing between uORF2 and the AUG^Elk1.</p

Analysis of the <i>ELK1</i> transcript in N2a cells.

<p>(A). RT-PCR amplification from total RNA isolated from N2a cells using primer sets in regions corresponding to the rat exon I and exon III. The experiment has been performed on duplicate samples and an RT minus control is included. (B). Alignment of the mouse, rat and human cDNA sequences up to the AUG^sElk1 for the 5′UTR^S. The uAUGs are indicated in red, the iAUGs in blue and Elk1/sElk1 AUGs in green. The small uORF2 is underlined. (C). A schematic representation indicating the positioning of the iAUGs in both mouse/rat and human transcripts. The Elk1 ORF is indicated above the line and the iAUG^a/b/c ORF below the line. The black rectangle in the mouse/rat Elk1 indicates the third ORF that can be expressed from the iAUG^d. The colour coding for the AUGs corresponds to that depicted in B. The sequence below the AUG^sElk1 demonstrates how this codon overlaps the UGA stop codon for the iAUG^a/b/c ORF. The small highly conserved uORF2 derived from uAUG2 is also depicted. It is the major source of reinitiating ribosomes that scan downstream of the AUG^Elk1<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102890#pone.0102890-Rahim1" target="_blank">[8]</a>.</p

Elk1 expression in rodent cells.

<p>(A). Upper Panel: PC12 cells were treated with increasing doses of NGF at low or high serum concentrations. Cell extracts were prepared 5 days after NGF treatment and analysed by immunoblotting with the anti-Elk1 Ab^SC. M^Elk and M^sElk are markers for these proteins generated by transiently over-expressing cDNA clones of each in HEK293T cells. Lower panel: RT-PCR was performed on total RNA extracted from PC12 cells cultured under the conditions indicated. Primers were positioned within the exon I and exon III. Amplicons corresponding to the 5′UTR^L and 5′UTR^S are indicated as is an actin control. (B). N2a cells were transfected with increasing doses of either a murine (0.1 nmoles, 0.2 nmoles and 0.3 nmoles/well) or human siRNA (0.2 nmoles and 0.3 nmoles/well) directed against the <i>ELK1</i> transcript. Cells were harvested 24 hrs post-transfection and analysed by immunoblotting using the Ab^SC. The lane C is the non-transfected control, the lane M contains protein markers for Elk1 and sElk1. The lower panel is a shorter exposure of the sElk1 band. (C). Multiple independent N2a cells extracts were immunoblotted and probed with the Ab^SC. The blot was stripped and re-probed with the Ab^Ab. Markers for Elk1 and sElk1 are indicated. (D). Cell extracts prepared from PC12 cells grown under different serum concentrations +/− NGF and N2a cells were analysed by immunoblotting using the Ab^Ab. Markers for Elk1 and sElk1 are indicated. (E). cDNA clones expressing Elk1^HA and sElk1^HA were transiently expressed in N2a cells. At 16 hrs post-transfection cells were treated with cycloheximide (10 µg/mL) and harvested at the times indicated (in hrs). Proteins were analysed by immunoblotting with the HA Ab. The bands were quantitated and the calculated half-life for each is indicated (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102890#pone.0102890.s001" target="_blank">Figure S1</a>). The * indicates HA-tagged products migrating faster than sElk1^HA.</p

Insulin and IGF1 receptors are essential for XX and XY gonadal differentiation and adrenal development in mice

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El catequista : Revista semanal aprobada y bendecida por el Excmo. e Ilmo. Sr. Obispo de la Diócesis: Año II Número 15 - 1907 abril 11

Copia digital. Madrid : Ministerio de Cultura. Subdirección General de Coordinación Bibliotecaria, 200

Transcriptional and post-transcriptional profile of human chromosome 21

Recent studies have demonstrated extensive transcriptional activity across the human genome, a substantial fraction of which is not associated with any functional annotation. However, very little is known regarding the post-transcriptional processes that operate within the different classes of RNA molecules. To characterize the post-transcriptional properties of expressed sequences from human chromosome 21 (HSA21), we separated RNA molecules from three cell lines (GM06990, HeLa S3, and SK-N-AS) according to their ribosome content by sucrose gradient fractionation. Polyribosomal-associated RNA and total RNA were subsequently hybridized to genomic tiling arrays. We found that ∼50% of the transcriptional signals were located outside of annotated exons and were considered as TARs (transcriptionally active regions). Although TARs were observed among polysome-associated RNAs, RT-PCR and RACE experiments revealed that ∼40% were likely to represent nonspecific cross-hybridization artifacts. Bioinformatics discrimination of TARs according to conservation and sequence complexity allowed us to identify a set of high-confidence TARs. This set of TARs was significantly depleted in the polysomes, suggesting that it was not likely to be involved in translation. Analysis of polysome representation of RefSeq exons showed that at least 15% of RefSeq transcripts undergo significant post-transcriptional regulation in at least two of the three cell lines tested. Among the regulated transcripts, enrichment analysis revealed an over-representation of genes involved in Alzheimer's disease (AD), including APP and the BACE1 protease that cleaves APP to produce the pathogenic beta 42 peptide. We demonstrate that the combination of RNA fractionation and tiling arrays is a powerful method to assess the transcriptional and post-transcriptional properties of genomic regions