Analyses of zebrafish and Xenopus oocyte maturation reveal conserved and diverged features of translational regulation of maternal cyclin B1 mRNA

<p>Abstract</p> <p>Background</p> <p>Vertebrate development relies on the regulated translation of stored maternal mRNAs, but how these regulatory mechanisms may have evolved to control translational efficiency of individual mRNAs is poorly understood. We compared the translational regulation and polyadenylation of the cyclin B1 mRNA during zebrafish and <it>Xenopus </it>oocyte maturation. Polyadenylation and translational activation of cyclin B1 mRNA is well characterized during <it>Xenopus </it>oocyte maturation. Specifically, <it>Xenopus </it>cyclin B1 mRNA is polyadenylated and translationally activated during oocyte maturation by proteins that recognize the conserved AAUAAA hexanucleotide and U-rich Cytoplasmic Polyadenylation Elements (CPEs) within cyclin B1 mRNA's 3'<b>U</b>n<b>T</b>ranslated <b>R</b>egion (3'<b>UTR</b>).</p> <p>Results</p> <p>The zebrafish cyclin B1 mRNA was polyadenylated during zebrafish oocyte maturation. Furthermore, the zebrafish cyclin B1 mRNA's 3'UTR was sufficient to stimulate translation of a reporter mRNA during zebrafish oocyte maturation. This stimulation required both AAUAAA and U-rich CPE-like sequences. However, in contrast to AAUAAA, the positions and sequences of the functionally defined CPEs were poorly conserved between <it>Xenopus </it>and zebrafish cyclin B1 mRNA 3'UTRs. To determine whether these differences were relevant to translation efficiency, we analyzed the translational activity of reporter mRNAs containing either the zebrafish or <it>Xenopus </it>cyclin B1 mRNA 3'UTRs during both zebrafish and <it>Xenopus </it>oocyte maturation. The zebrafish cyclin B1 3'UTR was quantitatively less effective at stimulating polyadenylation and translation compared to the <it>Xenopus </it>cyclin B1 3'UTR during both zebrafish and <it>Xenopus </it>oocyte maturation.</p> <p>Conclusion</p> <p>Although the factors that regulate translation of maternal mRNAs are highly conserved, the target sequences and overall sequence architecture within the 3'UTR of the cyclin B1 mRNA have diverged to affect translational efficiency, perhaps to optimize levels of cyclin B1 protein required by these different species during their earliest embryonic cell divisions.</p

Generation of ribosome imprinted polymers for sensitive detection of translational responses

Whilst the profiling of the transcriptome and proteome even of single-cells becomes feasible, the analysis of the translatome, which refers to all messenger RNAs (mRNAs) engaged with ribosomes for protein synthesis, is still an elaborate procedure requiring millions of cells. Herein, we report the generation and use of “smart materials”, namely molecularly imprinted polymers (MIPs) to facilitate the isolation of ribosomes and translated mRNAs from merely 1,000 cells. In particular, we show that a hydrogel-based ribosome imprinted polymer could recover ribosomes and associated mRNAs from human, simian and mice cellular extracts, but did not selectively enrich yeast ribosomes, thereby demonstrating selectivity. Furthermore, ribosome imprinted polymers enabled the sensitive measurement of an mRNA translational regulatory event, requiring 1,000-fold less cells than current methodologies. These results provide first evidence for the suitability of MIPs to selectively recover ribonucleoprotein complexes such as ribosomes, founding a novel means for sensitive detection of gene regulation

Cytoplasmic Prep1 Interacts with 4EHP Inhibiting Hoxb4 Translation

embryo development. Interestingly, Prep1 contains a putative binding motif for 4EHP, which may reflect a novel unknown function. development effect. mRNA to the 5′ cap structure. This is the first demonstration that a mammalian homeodomain transcription factor regulates translation, and that this function can be possibly essential for the development of female germ cells and involved in mammalian zygote development

The Nanos3-3′UTR Is Required for Germ Cell Specific NANOS3 Expression in Mouse Embryos

BACKGROUND: The regulation of gene expression via a 3' untranslated region (UTR) plays essential roles in the discrimination of the germ cell lineage from somatic cells during embryogenesis. This is fundamental to the continuation of a species. Mouse NANOS3 is an essential protein required for the germ cell maintenance and is specifically expressed in these cells. However, the regulatory mechanisms that restrict the expression of this gene in the germ cells is largely unknown at present. METHODOLOGY/PRINCIPAL FINDINGS: In our current study, we show that differences in the stability of Nanos3 mRNA between germ cells and somatic cells is brought about in a 3'UTR-dependent manner in mouse embryos. Although Nanos3 is transcribed in both cell lineages, it is efficiently translated only in the germ lineage. We also find that the translational suppression of NANOS3 in somatic cells is caused by a 3'UTR-mediated mRNA destabilizing mechanism. Surprisingly, even when under the control of the CAG promoter which induces strong ubiquitous transcription in both germ cells and somatic cells, the addition of the Nanos3-3'UTR sequence to the coding region of exogenous gene was effective in restricting protein expression in germ cells. CONCLUSIONS/SIGNIFICANCE: Our current study thus suggests that Nanos3-3'UTR has an essential role in translational control in the mouse embryo

Synthesis of two SAPAP3 isoforms from a single mRNA is mediated via alternative translational initiation

In mammalian neurons, targeting and translation of specific mRNAs in dendrites contribute to synaptic plasticity. After nuclear export, mRNAs designated for dendritic transport are generally assumed to be translationally dormant and activity of individual synapses may locally trigger their extrasomatic translation. We show that the long, GC-rich 5′-untranslated region of dendritic SAPAP3 mRNA restricts translation initiation via a mechanism that involves an upstream open reading frame (uORF). In addition, the uORF enables the use of an alternative translation start site, permitting synthesis of two SAPAP3 isoforms from a single mRNA. While both isoforms progressively accumulate at postsynaptic densities during early rat brain development, their levels relative to each other vary in different adult rat brain areas. Thus, alternative translation initiation events appear to regulate relative expression of distinct SAPAP3 isoforms in different brain regions, which may function to influence synaptic plasticity

Allellic variants in regulatory regions of cyclooxygenase-2: association with advanced colorectal adenoma

Cyclooxygenase 2 (Cox-2) is upregulated in colorectal adenomas and carcinomas. Polymorphisms in the Cox-2 gene may influence its function and/or its expression and may modify the protective effect of nonsteroidal anti-inflammatory drugs (NSAIDs), thereby impacting individuals' risk of developing colorectal cancer and response to prevention/intervention strategies. In a nested case–control study, four polymorphisms in the Cox-2 gene (two in the promoter, −663 insertion/deletion, GT/(GT) and −798 A/G; one in intron 5-5229, T/G; one in 3′untranslated region (UTR)-8494, T/C) were genotyped in 726 cases of colorectal adenomas and 729 age- and gender-matched controls in the prostate, lung, colorectal, and ovarian (PLCO) cancer screening trial. There was no significant association between the Cox-2 polymorphisms and adenoma development in the overall population. However, in males, the relatively rare heterozygous genotype GT/(GT) at −663 in the promoter and the variant homozygous genotype G/G at intron 5-5229 appeared to have inverse associations (odds ratio (OR)=0.59, confidence interval (CI): 0.34–1.02 and OR=0.48, CI: 0.24–0.99, respectively), whereas the heterozygous genotype T/C at 3′UTR-8494 had a positive association (OR=1.31, CI: 1.01–1.71) with adenoma development. Furthermore, the haplotype carrying the risk-conferring 3′UTR-8494 variant was associated with a 35% increase in the odds for adenoma incidence in males (OR=1.35, CI: 1.07–1.70), but the one with a risk allele at 3′UTR-8494 and a protective allele at intron 5-5229 had no effect on adenoma development (OR=0.85, CI: 0.66–1.09). Gender-related differences in adenoma risk were also noted with tobacco usage and protective effects of NSAIDs. Our analysis underscores the significance of the overall allelic architecture of Cox-2 as an important determinant for risk assessment

Aberrant over-expression of a forkhead family member, FOXO1A, in a brain tumor cell line

<p>Abstract</p> <p>Background</p> <p>The mammalian FOXO (forkhead box, O subclass) proteins are a family of pleiotropic transcription factors involved in the regulation of a broad range of cellular processes critical for survival. Despite the essential and diverse roles of the FOXO family members in human cells and their involvement in tumor pathogenesis, the regulation of <it>FOXO </it>expression remains poorly understood. We have addressed the mechanisms underlying the high level of expression of the <it>FOXO1A </it>gene in a cell line, PER-453, derived from a primitive neuroectodermal tumor of the central nervous system (CNS-PNET).</p> <p>Methods</p> <p>The status of the <it>FOXO1A </it>locus in the PER-453 CNS-PNET cell line was investigated by Southern blotting and DNA sequence analysis of the proximal promoter, 5'-UTR, open reading frame and 3'-UTR. FOXO1A expression was assessed by conventional and quantitative RT-PCR, Northern and Western blotting.</p> <p>Results</p> <p>Quantitative real-time RT-PCR (qRT-PCR) data indicated that after normalization to <it>ACTB </it>mRNA levels, canonical <it>FOXO1A </it>mRNA expression in the PER-453 cell line was 124-fold higher than the average level of five other CNS-PNET cell lines tested, 24-fold higher than the level in whole fetal brain, and 3.5-fold higher than the level in fetal brain germinal matrix cells. No mutations within the <it>FOXO1A </it>open reading frame or gross rearrangements of the <it>FOXO1A </it>locus were detected. However, a single nucleotide change within the proximal promoter and several nucleotide changes within the 3'-UTR were identified. In addition, two novel <it>FOXO1A </it>transcripts were isolated that differ from the canonical transcript by alternative splicing within the 3'-UTR.</p> <p>Conclusion</p> <p>The CNS-PNET cell line, PER-453, expresses <it>FOXO1A </it>at very high levels relative to most normal and cancer cells from a broad range of tissues. The <it>FOXO1A </it>open reading frame is wild type in the PER-453 cell line and the abnormally high <it>FOXO1A </it>mRNA expression is not due to mutations affecting the 5'-UTR or proximal promoter. Over expression of <it>FOXO1A </it>may be the result of PER-453 specific epimutations or imbalances in regulatory factors acting at the promoter and/or 3'-UTR.</p

RASOnD - A comprehensive resource and search tool for RAS superfamily oncogenes from various species

<p>Abstract</p> <p>Background</p> <p>The Ras superfamily plays an important role in the control of cell signalling and division. Mutations in the Ras genes convert them into active oncogenes. The Ras oncogenes form a major thrust of global cancer research as they are involved in the development and progression of tumors. This has resulted in the exponential growth of data on Ras superfamily across different public databases and in literature. However, no dedicated public resource is currently available for data mining and analysis on this family. The present database was developed to facilitate straightforward accession, retrieval and analysis of information available on Ras oncogenes from one particular site.</p> <p>Description</p> <p>We have developed the RAS Oncogene Database (RASOnD) as a comprehensive knowledgebase that provides integrated and curated information on a single platform for oncogenes of Ras superfamily. RASOnD encompasses exhaustive genomics and proteomics data existing across diverse publicly accessible databases. This resource presently includes overall 199,046 entries from 101 different species. It provides a search tool to generate information about their nucleotide and amino acid sequences, single nucleotide polymorphisms, chromosome positions, orthologies, motifs, structures, related pathways and associated diseases. We have implemented a number of user-friendly search interfaces and sequence analysis tools. At present the user can (i) browse the data (ii) search any field through a simple or advance search interface and (iii) perform a BLAST search and subsequently CLUSTALW multiple sequence alignment by selecting sequences of Ras oncogenes. The Generic gene browser, GBrowse, JMOL for structural visualization and TREEVIEW for phylograms have been integrated for clear perception of retrieved data. External links to related databases have been included in RASOnD.</p> <p>Conclusions</p> <p>This database is a resource and search tool dedicated to Ras oncogenes. It has utility to cancer biologists and cell molecular biologists as it is a ready source for research, identification and elucidation of the role of these oncogenes. The data generated can be used for understanding the relationship between the Ras oncogenes and their association with cancer. The database updated monthly is freely accessible online at <url>http://202.141.47.181/rasond/</url> and <url>http://www.aiims.edu/RAS.html</url>.</p

Diversity of Protein and mRNA Forms of Mammalian Methionine Sulfoxide Reductase B1 Due to Intronization and Protein Processing

Background: Methionine sulfoxide reductases (Msrs) are repair enzymes that protect proteins from oxidative stress by catalyzing stereospecific reduction of oxidized methionine residues. MsrB1 is a selenocysteine-containing cytosolic/nuclear Msr with high expression in liver and kidney. Principal Findings: Here, we identified differences in MsrB1 gene structure among mammals. Human MsrB1 gene consists of four, whereas the corresponding mouse gene of five exons, due to occurrence of an additional intron that flanks the stop signal and covers a large part of the 3′-UTR. This intron evolved in a subset of rodents through intronization of exonic sequences, whereas the human gene structure represents the ancestral form. In mice, both splice forms were detected in liver, kidney, brain and heart with the five-exon form being the major form. We found that both mRNA forms were translated and supported efficient selenocysteine insertion into MsrB1. In addition, MsrB1 occurs in two protein forms that migrate as 14 and 5 kDa proteins. We found that each mRNA splice form generated both protein forms. The abundance of the 5 kDa form was not influenced by protease inhibitors, replacement of selenocysteine in the active site or mutation of amino acids in the cleavage site. However, mutation of cysteines that coordinate a structural zinc decreased the levels of 5 and 14 kDa forms, suggesting importance of protein structure for biosynthesis and/stability of these forms. Conclusions: This study characterized unexpected diversity of protein and mRNA forms of mammalian selenoprotein MsrB1

Identification of Essential Sequences for Cellular Localization in BRMS1 Metastasis Suppressor

10 páginas, 5 figuras. PMID: 19649328 [PubMed] PMCID: PMC2713406BACKGROUND: Breast cancer metastasis suppressor 1 (BRMS1) reduces the number and the size of secondary tumours in a mouse model without affecting the growth of the primary foci upon its re-expression. Knockdown of BRMS1 expression associates with metastasis. The molecular details on BRMS1 mechanism of action include its ability to function as a transcriptional co-repressor and consistently BRMS1 has been described as a predominantly nuclear protein. Since cellular distribution could represent a potential mechanism of regulation, we wanted to characterize BRMS1 sequence motifs that might regulate its cellular distribution. According to its amino acids sequence, BRMS1 contain two putative nuclear localization signals, however none of them has been proved to work so far. METHODOLOGY/PRINCIPAL FINDINGS: By using well known in vivo assays to detect both nuclear import and export signal, we have characterized, in the present study, one functional nuclear localisation signal as necessary and sufficient to promote nuclear transport. Additionally, the outcome of a directed yeast two-hybrid assay identify importin alpha6 as a specific partner of BRMS1 thus speculating that BRMS1 nuclear import could be specifically mediated by the reported nuclear transporter. Besides, the combination of a computational searching approach along the utilization of a nuclear export assay, identified a functional motif within the BRMS1 sequence responsible for its nuclear export, that resulted not affected by the highly specific CRM1 inhibitor Leptomycin-B. Interspecies heterokaryon assay demonstrate the capability of BRMS1 to shuttle between the nuclear and cytosolic compartments CONCLUSIONS/SIGNIFICANCE: Our results show for the first time that BRMS1 contains both nuclear import and export signals enabling its nucleo-cytoplasmic shuttling. These findings contributes new data for the understanding of the BRMS1 functions and allow us to speculate that this phenomenon could represent a novel mechanism for regulating the activity of BRMS1 or its associated cytosolic partnersThis work was supported by Spanish Ministerio de Ciencia y Tecnología (Grant SAF2006-10269), Ministerio de Ciencia e Innovación (Grant SAF2008-04048-E) and by a grant from Fundación Mutua Madrileña.Peer reviewe