The context organization of functional regions in yeast genes with high-level expression

With the example of yeast genes, context organization was compared for functional gene regions (promoter, 5'-UTR, 3'-UTR) and tested for association with the level of gene expression. Several parameters (nucleotide composition, dinucletoide content bias) proved to correlate with expression level, each functional region having its specific features. Context optimization of a functional region was assumed to be essential for highly efficient interaction with the expression system of the cell. Specific context features were considered as dispersed signals important for high-level gene expression.     

The Expression of Vasoactive Intestinal Peptide Receptor 1 Is Negatively Modulated by MicroRNA 525-5p

Background

The human Vasoactive Intestinal Peptide (VIP) is a neurokine with effects on the immune system where it is involved in promoting tolerance. In this context, one of its receptors, VPAC1, has been found to be down-modulated in cells of the immune network in response to activating stimuli. In particular, the bacterial liposaccaride (LPS), a strong activator of the innate immune system, induces a rapid decrease of VPAC1 expression in monocytes and this event correlates with polymorphisms in the 3′-UTR of the gene.

Methodology/Principal Findings

MicroRNA 525-5p, having as putative target the 3′-UTR region of VPAC1, has been analysed for its expression in monocytes and for its role in down-modulating VPAC1 expression. We report here that miR-525-5p is promptly up-regulated in LPS-treated monocytes. This microRNA, when co-transfected in 293T cells together with a construct containing the 3′-UTR of the VPAC1 gene, significantly reduced the luciferase activity in a standard expression assay. The U937 cell line as well as primary monocytes enforced to express miR-525-5p, both down-modulate VPAC1 expression at similar extent.

Conclusions/Significance

Our results show that the response to an inflammatory stimulus elicits in monocytes a rapid increase of miR-525-5p that targets a signaling pathway involved in the control of the immune homeostasis.     

Contextual Features of Higher Plant mRNA 5"-Untranslated Regions

Computer analysis of nucleotide sequences of 5"-untranslated regions (5"-UTR) of higher plant mRNA adopted from the EMBL nucleotide sequence database was carried out. It was demonstrated that the average nucleotide frequencies of the leader sequences and adjacent regions of basal promoters are similar, whereas introns and 3"-UTR have a higher content of T and a lower content of C. A particular 5"-UTR contextual feature is a misbalance in the content of complementary nucleotides, probably caused by negative influence of the stable secondary structure on the translation properties of the leader sequence. Approximately 20% of 5"-UTR possess AUG triplets, i.e., twice as much as it has been estimated earlier. The properties of the open reading frames of the leader sequence (uORF) and presumable causes of their high content in 5"-UTR of eukaryotic mRNAs are discussed. The nature of correlation between some features of uORFs and protein-coding gene sequences is analyzed. It is demonstrated that in effectively translated mRNAs the leader AUG triplets are more frequently located in a nonoptimal context, whereas the terminating codons of uORFs more frequently exist in the optimal one. A hypothesis is put forward that the efficiency of termination at the uORF stop codon might substantially interfere with the mRNA translation activity.     

Regularory elements of the <Emphasis Type="Italic">copia</Emphasis> retrotransposon determine different levels of expression in different organs of males and females of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Using a model system in which the expression of the reporter gene lacZ is under the control of five deleted variants of the copia retrotransposon regulatory region, which includes the 5′-long terminal repeat (LTR) and the 5′-untranslated region (5′-UTR), their contribution to the control of retrotransposon activity in different organs of males and females of Drosophila melanogaster was analyzed. The whole regulatory region provides expression of the reporter gene at the embryonic stage, and in larvae and adult flies only in generative organs. The 5′-end of LTR harbors a positive regulator that determines expression of the retrotransposon in organs of all types. The 3′-end of LTR harbors a negative regulator, which is sex- and time-specific: it represses copia expression in generative organs of males at all stages of development, and only at the imaginal stage in somatic tissues, without any effect on the expression of the retrotransposon in females. 5′-UTR contains a negative regulator of copia expression: it decreases the expression in embryos and generative organs and blocks it in somatic tissues. It may be suggested that a complex set of regulatory elements was formed in the course of the evolution of the retrotransposon, which made it possible to maintain a certain level of its expression in different types of cells and tissues and at different stages of development and, thus, to limit the harm caused to the host and provide the possibility for the retrotransposon to exist in the host genome over many generations.     

真核mRNA的3′非翻译区转录后水平调控作用研究进展

真核生物mRNA的3′非翻译区(3′_UTR)在基因表达的转录后调控中起着重要作用:3′_UTR内存在末端加工信号以指导mRNA3′末端的加工;3′_UTR不但控制mRNA的稳定性及降解速率、协助辨认特殊密码子,而且还控制着mRNA的翻译时间、位点及控制其翻译起始及效率等。     

Limitation in use of luciferase reporter genes for 3′-untranslated region analysis

Luciferase reporter genes are widely used for the functional characterization of regulatory elements in 3′-untranslated region (3′-UTR). Using a transient expression assay system with pancreatic cell lines, we demonstrated that luciferase reporter gene constructs show not only the elements with special sequences in 3′-UTR that can affect luciferase activity, but also elements containing random sequences that were ligated into the same site. The extent of the decrease in luciferase activity was dependent on the length of the DNA fragments. Our findings strongly suggested a need to re-examine the 3′-UTR characterizations of many eukaryotic genes which have been studied to date with luciferase reporter genes. Lintao Wang and Jingjing Zhang are equal contributors.     

Genetic variations of mitochondrial antiviral signaling gene (MAVS) in domestic chickens

Mitochondrial antiviral signaling (MAVS) gene plays a key role in antiviral regulation in mammals potentially by activating IRF3/7 and NF-κB and leading to the induction of type I interferon (IFN)-mediated antiviral and inflammatory responses. In this study, we screened genetic polymorphisms of the MAVS gene in various Chinese domestic chicken breeds/populations and evaluated its potential effect on gene expression. Among the sequenced fragment (4678 bp), a total of 75 single nucleotide polymorphisms (SNPs) were identified in 46 chickens from 10 breeds/populations, including 30 coding SNPs and 45 non-coding SNPs. Extremely high haplotype diversity (37 nucleotide haplotypes, 18 amino acid haplotypes) was observed in the coding region (CDS), and a similar pattern of high polymorphisms was also observed for the 3′-untranslated region (3′-UTR). Luciferase assays of two representative 3′-UTR haplotypes were performed in both HEK293 cells and DF-1 chicken fibroblast cells, and we found that they were differentially associated with different abilities on regulating mRNA expression level (P < 0.05). Collectively, we observed a considerably high genetic variability of the MAVS gene, and the 3′-UTR variants had an ability to regulate mRNA expression. These results would cast some clues on understanding the potential role of MAVS on viral resistance in chicken.     

Molecular cloning of a dehydration-responsive protein gene (MRD22) from mulberry,and determination of abiotic stress patterns of MRD22 gene expression

A full-length cDNA sequence coding for dehydration-responsive protein gene of mulberry tree, which we designated was MRD22 (GenBank accession number: JQ804833) was cloned based on mulberry expressed sequence tags (ESTs). MRD22 is 1503 bp long, contains a 334 bp 5′-UTR (untranslated region) and a 563 bp 3′-UTR, encodes 201 amino acids with a predicted molecular weight of 54.28 kDa and an isoelectric point of 9.35. Phylogenetic analysis based on MRD22 sequences from different species showed that mulberry has close relationship with Populus trichocarpa, Ricinus communis, Camellia sinensis, Gossypium hirsutum, Gossypium barbadense and so on. The expression level of the MRD22 gene under conditions of drought, low temperature and salt stresses was quantified by qRT-PCR. The results show that the expression level changed significantly under the stress conditions compared to the normal growth environment. It helps us to get a better understanding of the molecular basis for signal transduction mechanisms underlying the stress response in mulberry.     

HuD Regulates the cpg15 Expression Via the 3′-UTR and AU-Rich Element

The candidate plasticity related gene 15 (cpg15) plays important roles in neural development and plasticity. In the present study, we studied the role of the cpg15 3′-untranslated region (UTR) in regulating the expression of the gene. The results showed that the presence of the 3′-UTR significantly decreases, while loss of a putative AU-rich element (ARE) in the 3′-UTR increases the cpg15 expression, indicating that the 3′-UTR and ARE may be essential for regulation of cpg15 expression. In addition, HuD, a neural-specific RNA binding protein, increased the cpg15 expression, which depends on the presence of the 3′-UTR and ARE. RNA-binding protein immunoprecipitation (RIP) assay demonstrated that HuD forms a complex with cpg15 mRNA in the cells of rat hippocampus. Deletion of HuD domains RRM1 plus RRM2 or Hinge region plus RRM3 attenuates the function of HuD in enhancing the cpg15 expression. The results suggest that HuD regulates the cpg15 expression via the 3′-UTR-mediated mechanism, which requires the presence of the ARE.