OBJECTIVETo analyze the alteration in alternative polyadenylation (APA) sites of tumor-related genes in gastric cancer cells.

METHODSWe used 3'RACE to capture the APA sites of two tumor-related genes (HSP90α and SEC11A) in gastric cancer cell lines MKN45, MKN28 and AGS, and compared the results with annotated poly(A) sites in UCSC database.

RESULTSWe found new APA sites in the two tumor-related genes in gastric cancer cells to produce new mRNA isoforms with different 3'UTRs.

CONCLUSIONSThere are new mRNA isoforms of HSP90α and SEC11A derived from ATA in gastric cancer cells, which provides new insights into the mechanisms of gastric tumorigenesis.

Cell Line, Tumor ; Cell Transformation, Neoplastic ; Genes, Neoplasm ; Humans ; Polyadenylation ; Stomach Neoplasms ; genetics

Varying length of messenger RNA (mRNA) 3′-untranslated region is generated by alternating the usage of polyadenylation sites during pre-mRNA processing. It is prevalent through all eukaryotes and has emerged as a key mechanism for controlling gene expression. Alternative polyadenylation (APA) plays an important role for cell growth, proliferation, and differentiation. In this review, we discuss the functions of APA related with various physiological conditions including cellular metabolism, mRNA processing, and protein diversity in a variety of disease models. We also discuss the molecular mechanisms underlying APA regulation, such as variations in the concentration of mRNA processing factors and RNA-binding proteins, as well as global transcriptome changes under cellular signaling pathway.
Eukaryota ; Gene Expression ; Humans* ; Metabolism ; Polyadenylation* ; RNA Precursors ; RNA, Messenger ; RNA-Binding Proteins ; TOR Serine-Threonine Kinases ; Transcriptome

OBJECTIVETo perform a genome-wide alternative polyadenylation (APA) profiling in both mouse female germline stem cells (FGSCs) and embryonic stem cells (ESCs) and explore the role of germline-specific APA in the biological behaviors of FGSCs.

METHODSWe used a high-throughput sequencing-based method 3T-Seq to profile the genome-wide 3' termini of the transcripts and delineate all the APA sites in mouse FGSCs and ESCs. The genes with altered APA sites in FGSCs compared with ESCs were analyzed with DAVID Gene Ontology tool for their biological roles.

RESULTSWe identified a total of 50243 APA sites in 16973 genes. In FGSCs, 1148 genes were shown to have alterations in 3'UTR length, among which 795 ( 66%) genes had shortened and 353 (34%) had lengthened 3'UTR. Some of the genes with shortened 3'UTR were involved in germ cell development.

CONCLUSIONSOur genome-wide APA profiling analysis reveals a cell type-specific APA alternation in FGSCs, and APA-mediated 3'UTR alteration contributes to germline-related biological process. This study provides a framework for understanding the post-transcriptional regulation mechanisms in FGSCs.

3' Untranslated Regions ; Animals ; Cell Differentiation ; Embryonic Germ Cells ; metabolism ; Embryonic Stem Cells ; metabolism ; Female ; Gene Expression Regulation ; Genome ; Mice ; Polyadenylation

Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the genus Arterivirus in the family Arteriviridae, is the most important viral pathogens in swine industry worldwide. Here, we have investigated 5' and 3' cis-acting RNA elements required for PRRSV genome replication. Using the infectious PRRSV cDNA, we have manipulated the genomic RNA to generate mutant genomic RNAs, transfected these mutants into susceptible MARC-145 cells, and examined the competence of RNA replication. We found three genetic factors that were essential for viral replication. First, the cap structure present at the 5'-end of the genome was absolutely required for RNA replication. Secondly, polyadenylation of the genomic RNA at the 3'-end was also essential for RNA replication. Thirdly, approximately 100-nucleotide region just upstream of the N protein-coding region was crucial for genomic RNA replication. Taken together, our findings indicate that replication of PRRSV genomic RNA requires three important cis-acting RNA elements: 5' cap structure, 3' poly(A) motif, and an internal sequence of about 100 nucleotides. Further investigation is needed to elucidate the molecular mechanism(s) of how these elements act on PRRSV genome replication.
Arteriviridae ; Arterivirus ; DNA, Complementary ; Genome ; Humans ; Mental Competency ; Nucleotides ; Polyadenylation ; Porcine Reproductive and Respiratory Syndrome* ; Porcine respiratory and reproductive syndrome virus* ; RNA* ; Swine

The cytoplasmic polyadenylation element (CPE)-binding protein (CPEB) binds to CPE containing mRNAs on their 3' untranslated regions (3'UTRs). This RNA binding protein comes out many important tasks, especially in learning and memory, by modifying the translational efficiency of target mRNAs via poly (A) tailing. Overexpressed CPEB has been reported to induce the formation of stress granules (SGs), a sort of RNA granule in mammalian cell lines. RNA granule is considered to be a potentially important factor in learning and memory. However, there is no study about RNA granule in Aplysia. To examine whether an Aplysia CPEB, ApCPEB1, forms RNA granules, we overexpressed ApCPEB1-EGFP in Aplysia sensory neurons. Consistent with the localization of mammalian CPEB, overexpressed ApCPEB1 formed granular structures, and was colocalized with RNAs and another RNA binding protein, ApCPEB, showing that ApCPEB1 positive granules are RNA-protein complexes. In addition, ApCPEB1 has a high turnover rate in RNA granules which were mobile structures. Thus, our results indicate that overexpressed ApCPEB1 is incorporated into RNA granule which is a dynamic structure in Aplysia sensory neuron. We propose that ApCPEB1 granule might modulate translation, as other RNA granules do, and furthermore, influence memory.
Animals ; Aplysia/genetics/*metabolism ; Fluorescence Recovery After Photobleaching ; RNA/genetics/metabolism ; Sensory Receptor Cells/*metabolism ; mRNA Cleavage and Polyadenylation Factors/genetics/metabolism/*physiology

The earliest stages of mammalian embryogenesis are governed by the activity of maternally inherited transcripts and proteins. Cytoplasmic polyadenylation of selected maternal mRNA has been reported to be a major control mechanism of delayed translation during preimplantation embryogenesis in mice. The presence of cis-elements required for cytoplasmic polyadenylation (e.g., CPE) can serve as a useful tag in the screening of maternal genes partaking in key functions in the transcriptionally dormant egg and early embryo. However, due to its relative simplicity, UA-rich sequences satisfying the canonical rule of known CPE consensus sequences are often found in the 3'-UTR of maternal transcripts that do not actually undergo cytoplasmic polyadenylation. In this study, we developed a method to confirm the validity of candidate CPE sequences in a given gene by a multiplex comparison of 3'-UTR sequences between mammalian homologs. We found that genes undergoing cytoplasmic polyadenylation tend to create a conserved block around the CPE, while CPE-like sequences in the 3'-UTR of genes lacking cytoplasmic polyadenylation do not exhibit such conservation between species. Through this cross-species comparison, we also identified an alternative CPE in the 3'-UTR of tissue-type plasminogen activator (tPA), which is more likely to serve as a functional element. We suggest that verification of CPEs based on sequence conservation can provide a convenient tool for mass screening of factors governing the earliest processes of mammalian embryogenesis.
Animals ; Consensus Sequence ; Cytoplasm ; Embryonic Development ; Embryonic Structures ; Female ; Mass Screening ; Mice ; Ovum ; Polyadenylation ; Pregnancy ; RNA, Messenger, Stored ; Tissue Plasminogen Activator

BACKGROUNDSurvivin is an oncoprotein silenced in normal mature tissues but reactivated in serous ovarian cancer (SOC). Although transcriptional activation is assumed for its overexpression, the long 3'-untranslated region (3'-UTR) in survivin gene, which contains many alternate polyadenylation (APA) sites, implies a propensity for posttranscriptional control and therefore was the aim of our study.

METHODSThe abundance of the coding region, the proximal and the distal region of survivin mRNA 3'-UTR, was evaluated by real-time polymerase chain reaction (PCR) in SOC samples, cell lines, and normal fallopian tube (NFT) tissues. The APA sites were confirmed by rapid amplification of cDNA 3' ends and DNA sequencing. Real-time PCR were used to screen survivin-targeting microRNAs (miRNAs) that were inversely correlated with survivin. The expression of an inversely correlated miRNA was restored by pre-miRNA transfection or induction with a genotoxic agent to test its inhibitory effect on survivin overexpression.

RESULTSVarying degrees of APA were observed in SOC by comparing the abundance of the proximal and the distal region of survivin 3'-UTR, and changes of 3'-UTR correlated significantly with survivin expression (r = 0.708, P< 0.01). The main APA sites are proved at 1197 and 1673 of survivin 3'-UTR by DNA sequencing. Higher level of 3'-UTR proximal region than coding region was observed in NFT, as well as in SOC and cell lines. Among the survivin-targeting miRNAs, only a few highly expressed miRNAs were inversely correlated with survivin levels, and they mainly targeted the distal part of the 3'-UTR. However, in ovarian cancer cells, restoration of an inversely correlated miRNA (miR-34c) showed little effect on survivin expression.

CONCLUSIONSIn NFT tissues, survivin is not transcriptionally silenced but regulate posttranscriptionally. In SOC, aberrant APA leads to the shortening of survivin 3'-UTR which enables it to escape the negative regulation of miRNAs and is responsible for survivin up-regulation.

3' Untranslated Regions ; genetics ; Female ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; metabolism ; MicroRNAs ; genetics ; Ovarian Neoplasms ; genetics ; metabolism ; Polyadenylation ; Real-Time Polymerase Chain Reaction

OBJECTIVETo verify if mutated polyadenylation signal retroviruses can produce viral-host readthrough transcripts (Rth) and have the ability to transform human gastric epithelial GES-1 cells, and to discuss the new functions of retroviruses in gastric cancer related gene research.

METHODSThe polyadenylation signal-deficient retrovirus vector mutated by PCR site-directed mutagenesis was used to make polyadenylation signal-deficient retroviruses by PA317 packaging cells. The GES-1 cells were infected by the viruses and selected by G418. Viral-host readthrough RNAs were checked by Northern blot. The cell growth and soft agar assay were run to test the transformed cells.

RESULTSpolyadenylation signal-deficient retroviruses could be packaged by PA317 packaging cells. The viruses had the ability to infect GES-1 cells. Northern blot analysis of viral RNA from infected pools and individual G418-resistant clones demonstrated that mutation of consensus LTR polyadenylation signals generated Rth viral RNA in the infected GES-1 cells. Phenotypic analysis results showed that the GES-1 cells infected with plyadenylation signal mutant viruses tended to grow in a cluster manner. Pools of PA317 cells infected with mutant viruses were able to form colonies in soft agar with a higher efficiency than control or uninfected cells.

CONCLUSIONHost readthrough transcripts generated by polyadenylation signal mutant viruses may contribute to transformation GES-1 cell phenotypes. The mutant vectors and the method described in the present work may be useful as tools to trap and identify genes involved in retroviral insertion mediated cell transformation.

Animals ; Cell Line ; Cell Transformation, Neoplastic ; Epithelial Cells ; cytology ; metabolism ; virology ; Fibroblasts ; cytology ; virology ; Humans ; Mice ; Mutagenesis, Site-Directed ; RNA 3' Polyadenylation Signals ; genetics ; RNA, Viral ; metabolism ; Retroviridae ; genetics ; Stomach ; cytology ; Terminal Repeat Sequences

Adult ; Benzamides ; pharmacology ; Humans ; Hypereosinophilic Syndrome ; drug therapy ; genetics ; Imatinib Mesylate ; Male ; Mutation ; Oncogene Proteins, Fusion ; Piperazines ; pharmacology ; Pyrimidines ; pharmacology ; Receptor, Platelet-Derived Growth Factor alpha ; genetics ; mRNA Cleavage and Polyadenylation Factors

OBJECTIVETo investigate the clinical and laboratory features of chronic eosinophilic leukemias (CEL) and hypereosinophilic syndrome (HES).

METHODSThe clinical manifestations, laboratory parameters were retrospectively analyzed in 20 patients with HES/CEL. Detection of the FIP1L1-PDGFRA fusion gene was performed by nested RT-PCR. JAK2 V617F mutation screening was processed through allele-specific PCR combined with sequence analysis. PCR-RFLP was used to discriminate homozygous from heterozygous mutation patterns. TCR gamma rearrangement was detected by PCR.

RESULTSOf the 20 patients, 19 were males and one female, with a median age of 33 (20 to 57) years. The FIP1L1-PDGFRA fusion gene positivity in bone marrow mononuclear cells in 12 cases was identified. All the breakpoints were identified by direct sequencing of cloned RT-PCR products in FIP1L1 intron 10 - 12 and in PDGFRA exon 12. In CEL the most common involved organs were lungs, heart and nervous system. Splenomegaly was significantly more frequent in CEL than in HES (92.5% vs 42.5%, P = 0.031). Anemia and myelofibrosis were common in CEL. There was no significant difference in circulating absolute eosinophil, leukocyte, platelet counts, hemoglobin level and percentages of eosinophil and blast cell in bone marrow between CEL and HES. The morphological abnormalities of eosinophils on bone marrow smear were easily found in CEL, including hypogranularity, and cytoplasmic vacuolization, increased basophilic granule. One patient with HES was found to have heterozygous JAK2 V617F mutation. Six patients had TCR gamma rearrangement, including 4 CEL and 2 HES.

CONCLUSIONS(1) There is a male predominance in HES/CEL, and the median age was in the thirties. (2) The most common involved organs in CEL were lung, heart and nervous system. Bone marrow morphology might be of a little help in diagnosis of CEL. (3) JAK2 V617F may be involved in the pathogenesis of HES. (4) Patients with CEL carried the FIP1L1-PDGFRA fusion gene and TCR gamma rearrangement concurrently, their relationship warrants further study.

Adult ; Female ; Gene Rearrangement ; Genes, T-Cell Receptor gamma ; genetics ; Humans ; Hypereosinophilic Syndrome ; diagnosis ; genetics ; Janus Kinase 2 ; genetics ; Male ; Middle Aged ; Mutation ; Receptor, Platelet-Derived Growth Factor alpha ; genetics ; Retrospective Studies ; Young Adult ; mRNA Cleavage and Polyadenylation Factors ; genetics