Ribotrap Analysis of Proteins Associated with FHL3 3'Untranslated Region in Glioma Cells

Objective To screen the proteins associated with four-and-a-half LIM domains 3(FHL3) 3' untranslated region(3'UTR) in glioma cells. Methods Western blot was adopted to detect the regulatory effect of poly(C)-binding protein 2(PCBP2) on FHL3. Biotin pull-down and sliver staining were employed to screen and verify the candidate binding proteins of FHL3 3'UTR. Then liquid chromatography-tandem mass spectrometry(LC-MS/MS) and molecule annotation system were used to identify and analyze the candidate binding proteins. Immunoprecipitation was conducted to study the interaction between PCBP2 and polypyrimidine tract-binding protein 1(PTBP1), a binding protein identified by LC-MS/MS. Results PCBP2 could bind to FHL3 mRNA 3'UTR-A and inhibited the expression of FHL3 in T98 G glioms cells. 22 candidate binding proteins were identified. Among them, there were 11 RNA binding proteins, including PCBP2. PTBP1 associated with FHL3 mRNA 3'UTR and interacted with PCBP2 protein. Conclusion PCBP2 and PTBP1 can both associate with FHL3 mRNA 3'UTR through forming a protein complex.

TYMS gene 5'-and 3'-untranslated region polymorphisms and risk of non-syndromic cleft lip and palate in an Indian population

Dear Editor： Increased homocysteine levels due to vitamin B6 or B12 deficiency or genetic defects in folate pathway genes are associated with an increased incidence of non-syndromic cleft lip with or without cleft palate （NSCLP）tlj. Thymidylate synthase （TS） is a folate-dependent enzyme that catalyzes methylation of 2＇-deoxyuridine-5＇-monophosphate （dUMP） to 2＇-deox- ythymidine-5＇-monophosphate （dTMP）, a rate-limiting step in DNA synthesis,

Molecular Epidemiological Analysis of Echovirus 19 Isolated From an Outbreak Associated With Hand, Foot, and Mouth Disease (HFMD) in Shandong Province of China

To elucidate the genetic characterization and molecular epidemiological features of Echovirus 19 （El9） isolates collected from an outbreak associated with hand, foot and mouth disease （HFMD） in Tai＇an city of Shandong Province of China from July to September, 2003. Methods Thirty seven Echovirus 19 isolates were isolated from stool specimens and throat swabs collected during the outbreak, then major capsid （VP1） genomic sequence was determined, and phylogenetic tree was done based on the VP1 sequences among these 37 and other El9 viruses deposited in the Genbank. Also a representative strain named CHN-SD03-TN12 was selected for sequencing of 5′-untranslated regions （5′-UTR）. Results The identity rate was about 98.9%-100% among all these 37 El9 viruses. The genetic relationships between these 37 El9 isolates and other strains reported were also depicted. The identity rate was about 78.4%-78.9% compared with El9 reference strain Burke. The substitutions in the sequence of 5′-UTR resulted in changes in the conjectural properties of 5′-UTR of El9 viruses. Condusion The genetic features of El9 viruses isolated during the outbreak in Shandong Province in 2003 may be associated with a genetic and antigenic drift that changes the virulence of the Shandong isolates, but the molecular changes in Shandong El9 viruses contributing to their phenotype remain to be further illuminated. However, the sequences described in this paper substantiate the changes taken place in capsid VPI and 5′UTR regions. These substitutions may contribute to their tropism and virulence, and play a significant role in pathogenesis and clinical manifestations of the disease.

Inhibition of host immune response in colorectal cancer:Human leukocyte antigen-G and beyond

Colorectal cancer (CRC) is one of the most diffuse cancers worldwide and is still a clinical burden. Increasing evidences associate CRC clinical outcome to immune contexture represented by adaptive immune cells. Their type, density and location are summarized in the Immune Score that has been shown to improve prognostic prediction of CRC patients. The non-classical MHC class I human leukocyte antigen-G (HLA-G), is a crucial tumor-driven immune escape molecule involved in immune tolerance. HLA-G and soluble counterparts are able to exert inhibitory functions by direct interactions with inhibitory receptors present on both innate cells such as natural killer cells, and adaptive immune cells as cytotoxic T and B lymphocytes. HLA-G may play a prominent role in CRC strategies to avoid host immunosurveillance. This review highlights the current knowledge on HLA-G contribution in CRC, in related inflammatory diseases and in other type of cancers and disorders. HLA-G genetic setting (specific haplotypes, genotypes and alleles frequencies) and association with circulating/soluble profiles was highlighted. HLA G prognostic and predictive value in CRC was investigated in order to define a novel prognostic immune biomarker in CRC.

Mutual regulation between microRNA-373 and methyl-CpGbinding domain protein 2 in hilar cholangiocarcinoma

AIM:To investigate the reciprocal modulation between microRNA(miRNA) and DNA methylation via exploring the correlation between miR-373 and methyl-CpGbinding domain protein(MBD)2.METHODS:MiR-373 expression was examined using the TaqMan miRNA assay.Methylation of miR-373 was investigated using methylation-specific polymerase chain reaction,and recruitment of methyl binding proteins was studied using the chromatin immunoprecipitation assay.Mutation analysis was conducted using the QuikChange Site-Directed Mutagenesis kit.The activity of miR-373 gene promoter constructs and targeting at MBD2-three prime untranslated region(3'UTR) by miR-373 were evaluated by a dual-luciferase reporter gene assay.RESULTS:In hilar cholangiocarcinoma,miR-373 decreased and was closely associated with poor cell differentiation,advanced clinical stage,and shorter survival.The promoter-associated CpG island of miR-373 gene was hypermethylated and inhibited expression of miR-373.MBD2 was up-regulated and enriched at the promoter-associated CpG island of miR-373.Methylation-mediated suppression of miR-373 required MBD2 enrichment at the promoter-associated CpG island,and miR-373 negatively regulated MBD2 expression through targeting the 3'UTR.CONCLUSION:MiR-373 behaves as a direct transcriptional target and negative regulator of MBD2 activity through a feedback loop of CpG island methylation.

A novel constitutive promoter and its downstream 5′ UTR derived from cotton(Gossypium spp.) drive high-level gene expression in stem and leaf tissues

The development of genetically modified crops requires new promoters and regulatory regions to achieve high gene ex- pression and/or tissue-specific expression patterns in plants. To obtain promoter sequences of plants with new properties, we analyzed the expression traits of the cotton （Gossypium hirsutum） translation elongation factor 1A gene family. The results showed that the GhEF1A8 gene is highly expressed in different organs of cotton plants, and showed much higher transcript levels in stems and leaves. Its promoter （GhEFIA1.7） and the 5＂ untranslated region （5＂ UTR）, comprising a regulatory region named PGhEFIA8, were isolated from cotton and studied in stably transformed tobacco plants. The regulatory region sequences were fused to the 13-glucuronidase （GUS） reporter gene to characterize its expression pattern in tobacco. Histochemical and fiuorometric GUS activity assays demonstrated that PGhEF1A8 could direct GUS gene expression in all tissues and organs in transgenic tobacco, including leaves, stems, flowers, and roots. The level of GUS activity in the leaves and stems was significantly higher than in cauliflower mosaic virus （CaMV） 35S promoter：：GUS plants, but as same as CaMV 35S promoter：：GUS plants in flower and root tissues. GUS expression levels decreased 2-10-fold when the 5＂ UTR was absent from PGhEF1A8. Deletion analysis of the PGhEFIA8 sequence showed that the region -647 to -323 might possess negative elements that repress transgene expression in tobacco plants. The results suggested that the GhEFIA8 regulation region may represent a practical choice to direct high-level constitutive expression of transgenes and could be a valuable new tool in plant genetic engineering.

Utilization of Alternative Polyadenylation Signals in the Novel Human Apoptosis-Inducing Gene hap

hap, a novel human apoptosis-inducing gene which can interact with another newly discovered apoptosis-inducing geneASY, was identified, by cloning its cDNAs from human lung cell line (WI-38) cDNA library. Two major mRNA species (1.8 and 2.7 kb in length, respectively) were previously identified by Northern blot analysis of poly(A)+ RNA from human multiple tissues using partialhap cDNA as a probe. In the present work, the molecular mechanism accounting for the generation of the twohap transcripts were investigated. The rapid amplification of cDNA 3′-ends (3′-RACE) technique and the sequential Southern blot analysis, in conjunction with the sequencing analysis demonstrated that the twohap transcripts derive from the alternative polyadenylation site selection: a AATAAA signal at position 1 528–1 533 nt for the 1.8 kbhap mRNA: and a AATAAA signal at position 2 375–2 380 nt for the 2.7 kbhap mRNA. Furthermore, a number of regulatory elements withinhap 3′-untranslated region (3′-UTR) were also examined.

Alternative polyadenylation of mRNA and its role in cancer

Alternative polyadenylation(APA)is a molecular process that generates diversity at the 3′end of RNA polymeraseⅡtranscripts from over 60%of human genes.APA is derived from the existence of multiple polyadenylation signals(PAS)within the same transcript,and results in the differential inclusion of sequence information at the 3′end.While APA can occur between two PASs allowing for generation of transcripts with distinct coding potential from a single gene,most APA occurs within the untranslated region(3′UTR)and changes the length and content of these non-coding sequences.APA within the 3′UTR can have tremendous impact on its regulatory potential of the mRNA through a variety of mechanisms,and indeed this layer of gene expression regulation has profound impact on processes vital to cell growth and development.Recent studies have particularly highlighted the importance of APA dysregulation in cancer onset and progression.Here,we review the current knowledge of APA and its impacts on mRNA stability,translation,localization and protein localization.We also discuss the implications of APA dysregulation in cancer research and therapy.

Identification and initial characterization of the 3＇ end of gene transcripts encoding putative members of the pheromone receptor subfamily in Lepidoptera

Semiochemicals, including pheromones and kairomones, used in pest man- agement programs reduce the need for chemical insecticides, and understanding their interactions with their membrane receptors may help make them more effective in the field. Identification of odorant receptors in the Lepidoptera has mainly been achieved us- ing bioinformatics to search DNA sequences generated by genome or expressed sequence tag （EST） sequencing projects. This study reports a rapid method to identify members of the pheromone receptor subfamily in Lepidoptera. Degenerate oligonucleotide primers were designed against a conserved amino acid sequence in the carboxyl terminus of known lepidopteran pheromone receptors, and the primers were used in a 3＇ rapid amplifica- tion of complementary DNA （cDNA） ends procedure. Polymerase chain reaction products generated from seven different lepidopteran species were TA cloned and sequenced. The cDNA sequences of 25 transcripts were determined to encode potential members of the pheromone receptor subfamily. These cDNAs ranged from 238 to 642 bp and encoded 49-54 amino acids of the carboxyl terminus. Analysis of the 3＇ untranslated region reveals that most of the transcripts contain multiple polyadenylation signal sequences, and in the case ofManduca sexta, an alternate polyadenylation signal appears to be used in transcript processing. The 3＇ untranslated region was also useful in determining unique receptors en- coded by transcripts having highly similar nucleotide and amino acid sequences. Overall, this technique provides a complementary method of pheromone receptor identification in EST sequencing projects, or can be used as a stand-alone method in conjunction with 5＇ rapid amplification of cDNA ends procedures.

vsx1 3' untranlated region-mediated translation difference at different developmental stages of goldfish embryos

Visual system homeobox-1 （vsxl） is important in retinal progenitor proliferation, differentiation, and function maintenance of bipolar cells in vertebrates. Recent study in Xenopus laevis has shown that vsxl 3＇ untranslated region （3＇ UTR） can mediate cell-specific translation of vsxl mRNA in the bipolar cells of the retinal inner nuclear layer （INL）. vsxl is also transcribed at the early developmental stages prior to eye formation and its spatiotemporal expression patterns are conserved in all the examined vertebrates. In order to determine whether the vsxl 3＇ UTR has a role in regulating the spatiotemporal expression of vsxl during early embryogenesis, we constructed a vsxl UTR-controlled green fluorescent protein （GFP） reporter gene system and examined the GFP expression pattern in goldfish, Carassius auratus, at different developmental stages, Our results indicated that both the vsxl 5＇ UTR and the vsxl 3＇ UTR remarkably repressed GFP expression at transcription level but did not regulate tissue-specific translation at early developmental stages. GFP protein was ubiquitously expressed in the embryos injected with GFP-sensors containing vsxl UTRs before 60 h post-fertilization （hpf）. From hatching stage （72 hpf） onwards, however, GFP protein was specifically expressed in the bipolar cells of the retinal 1NL in the vsxl 3＇UTR-GFP-sensor embryos, but was still ubiquitously expressed in the embryos injected with GFP-sensor lacking vsxl 3＇ UTR. These observations showed a significant difference of vsxl 3＇ UTR-mediated translation between early and late developmental stages and suggested that vsxl 3＇ UTR might not be involved in regulating the spatiotemporal expression of vsxl until hatching stage during embryogenesis.