mTOR-coordinated Post-Transcriptional Gene Regulations: from Fundamental to Pathogenic Insights

Post-transcriptional regulations of mRNA transcripts such as alternative splicing and alternative polyadenylation can affect the expression of genes without changing the transcript levels. Recent studies have demonstrated that these post-transcriptional events can have significant physiological impacts on various biological systems and play important roles in the pathogenesis of a number of diseases, including cancers. Nevertheless, how cellular signaling pathways control these post-transcriptional processes in cells are not very well explored in the field yet. The mammalian target of rapamycin complex 1 (mTORC1) pathway plays a key role in sensing cellular nutrient and energy status and regulating the proliferation and growth of cells by controlling various anabolic and catabolic processes. Dysregulation of mTORC1 pathway can tip the metabolic balance of cells and is associated with a number of pathological conditions, including various types of cancers, diabetes, and cardiovascular diseases. Numerous reports have shown that mTORC1 controls its downstream pathways through translational and/or transcriptional regulation of the expression of key downstream effectors. And, recent studies have also shown that mTORC1 can control downstream pathways via post-transcriptional regulations. In this review, we will discuss the roles of post-transcriptional processes in gene expression regulations and how mTORC1-mediated post-transcriptional regulations contribute to cellular physiological changes. We highlight post-transcriptional regulation as an additional layer of gene expression control by mTORC1 to steer cellular biology. These emphasize the importance of studying post-transcriptional events in transcriptome datasets for gaining a fuller understanding of gene expression regulations in the biological systems of interest.

Antisense transcription regulates the expression of sense gene via alternative polyadenylation

Natural antisense transcripts (NAT) and alternative polyadenylation (APA) of messenger RNA (mRNA) are important contributors of transcriptome complexity, each playing a critical role in multiple biological processes. However, whether they have crosstalk and function collaboratively is unclear. We discovered that APA enriched in human sense-antisense (S-AS) gene pairs, and finally focused on RNASEH2C-KAT5 S-AS pair for further study. In cis but not in trans over-expression of the antisense KAT5 gene promoted the usage of distal polyA (pA) site in sense gene RNASEH2C, which generated longer 3' untranslated region (3'UTR) and produced less protein, accompanying with slowed cell growth. Mechanistically, elevated Pol II occupancy coupled with SRSF3 could explain the higher usage of distal pA site. Finally, NAT-mediated downregulation of sense gene's protein level in RNASEH2C-KAT5 pair was specific for human rather than mouse, which lacks the distal pA site of RNASEH2C. We provided the first evidence to support that certain gene affected phenotype may not by the protein of its own, but by affecting the expression of its overlapped gene through APA, implying an unexpected view for understanding the link between genotype and phenotype.

Alternative Polyadenylation in Human Diseases

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.

Aberrant Alternative Polyadenylation is Responsible for Survivin Up-regulation in Ovarian Cancer / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Survivin 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>Varying 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.</p><p><b>CONCLUSIONS</b>In 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.</p>

Genome-wide profiling of alternative polyadenylation in mouse female germline stem cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>We 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.</p><p><b>RESULTS</b>We 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.</p><p><b>CONCLUSIONS</b>Our 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.</p>

Depression help-seeking attitudes and behaviors among an Internet-based sample of Spanish-speaking perinatal women / Actitudes y comportamientos de búsqueda de ayuda para la depresión en una muestra basada en internet de mujeres de habla hispana en período perinatal

OBJECTIVE: To examine attitudes and beliefs related to help-seeking for depression among an international sample of pregnant women, a majority of whom were Spanish-speakers residing in Latin America. METHODS: More than 6 000 (n = 6 672) pregnant women met eligibility criteria and consented to participate between 15 January 2009-12 August 2011. Of these, 1 760 with a Latino/Hispanic background completed a baseline survey as part of a larger study. Group comparisons analyzed attitudes and behaviors related to seeking help for depression, while a logistic regression was conducted to identify demographic characteristics related to help-seeking support. RESULTS: Of the participants, three-fourths reported experiencing depression during or after their current or past pregnancies. The majority of participants did not seek help, and generally reported ambivalence about their depressive symptoms and uncertainty as to the helpfulness of others. However, 44.8% did seek help, mostly by speaking to family or partners and reported feeling fear, shame, and embarrassment about their symptoms. A current major depressive episode and an income less than or equal to US$ 10 000 were significant predictors of help-seeking behaviors. CONCLUSIONS: Data from this study suggest that when feeling sad or depressed, perinatal Latinas tend to seek emotional support first from family and friends and may underutilize mental health services when needed. The Internet is an effective means for reaching perinatal women, especially those in areas of the world where there may be barriers to accessing psychological resources.

OBJETIVO: Analizar las actitudes y las creencias relacionadas con la búsqueda de ayuda para la depresión en una muestra internacional de mujeres embarazadas, la mayor parte de ellas hispanohablantes y residentes en América Latina. MÉTODOS: Más de 6 000 mujeres embarazadas (n = 6 672) cumplieron los criterios de selección y aceptaron participar entre el 15 de enero del 2009 y el 12 de agosto del 2011. De estas, 1 760 de origen latino o hispano completaron una encuesta básica que formaba parte de un estudio más amplio. Mediante comparaciones de grupo, se analizaron las actitudes y los comportamientos relacionados con la búsqueda de ayuda para la depresión, mientras que, mediante regresión logística, se determinaron las características demográficas relacionadas con la búsqueda de ayuda o apoyo. RESULTADOS: De todas las participantes, tres cuartas partes notificaron sentimientos de depresión durante o después de los embarazos actuales o pasados. La mayor parte de ellas no buscaron ayuda, y en general manifestaron ambivalencia acerca de sus síntomas depresivos e incertidumbre en cuanto a la capacidad de ayuda de otras personas. Sin embargo, 44,8% buscaron ayuda, principalmente hablando con familiares o compañeros, y notificaron sentimientos de temor, culpabilidad y vergüenza acerca de sus síntomas. Un episodio depresivo mayor actual y unos ingresos iguales o inferiores a US$ 10 000 fueron factores predictivos significativos de comportamientos de búsqueda de ayuda. CONCLUSIONES: Los datos de este estudio indican que, cuando se sienten tristes o deprimidas, las mujeres latinas en período perinatal tienden a buscar en primer lugar el apoyo emocional de la familia y los amigos, y podrían subutilizar los servicios de salud mental cuando son necesarios. La internet es un medio eficaz para llegar a las mujeres en período perinatal, especialmente a las que viven en zonas del mundo donde pueden existir barreras para el acceso a los recursos psicológicos.

Identificação e estudo de expressão de variantes de poliadenilação e de microRNAs através de sequenciamento em larga escala em amostras de tumor colorretal / Identification and expression study of polyadenylation variants and microRNAs through large-scale sequencing in samples from colorectal tumor

A maioria dos mRNAs de eucariotos adquire uma cauda de poliA não codificadora na extremidade 3' durante a maturação, em um processo chamado de poliadenilação (poliA). Mais da metade dos genes de mamíferos apresentam múltiplos sítios de poliA, que podem levar à formação de transcritos com tamanhos variáveis da 3’ UTR. A poliA alternativa (APA) pode influenciar a localização, estabilidade e transporte dos transcritos, de maneira tecido-específica ou até mesmo doença-específica. Isto é resultante de muitos elementos em cis situados nas 3' UTRs estarem envolvidos na regulação pós-transcricional, tais como os sítios de ligação a miRNAs. Neste trabalho foi utilizado o sequenciamento de última geração para identificar e avaliar a expressão de variantes de poliA, e ainda correlacioná-las à expressão de miRNAs presentes nas mesmas amostras. Para tanto, um protocolo original de construção de bibliotecas de cDNAs enriquecidas para a extremidade 3’ dos transcritos foi desenvolvido, permitindo o sequenciamento preferencial de 3' UTRs e a identificação de sítios de poliA em duas linhagens derivadas de tumor colorretal (HCT116 e SW480). Variantes de poliA com 3' UTR mais curtas do que o transcrito referência (RefSeq com maior 3' UTR) foram identificadas para mais de 4.000 genes, correspondendo a cerca de 30% dos genes expressos nestas células. Cerca de 60% das variantes curtas perderam pelo menos um sítio alvo de miRNA. Especificamente para estes genes foi observada uma diminuição da razão da expressão da variante longa pela expressão da variante curta (razão VL/VC) em relação aos genes que não possuíam sítios alvo. Além disso, foi encontrada uma diferença significativa da razão VL/VC entre os genes que possuíam sítios alvo para miRNAs não expressos e genes que possuíam sítios alvo para miRNAs expressos. Essa redução foi ainda maior para os genes nos quais a variante curta perde sítios para miRNAs altamente expressos comparando-se com genes alvo de miRNAs de baixa...

Most eukaryotic mRNAs acquire an uncoded polyA tail at their 3' end during maturation in a process called polyadenylation (polyA). More than half of mammalian genes have multiple polyA sites, which can lead to the formation of variant transcripts with different 3' UTRs. Alternative polyA (APA) may influence location, stability and transport of transcripts, in tissue or diseasespecific manner. This results from many cis-acting elements located within the 3' UTRs being involved in post-transcriptional regulation, such as the miRNA binding sites. Here we used next generation sequencing to identify and evaluate the expression of polyA variants and also correlate them with miRNA expression in the same samples. To do so, an original cDNA library protocol to enrich for the transcripts’ 3' ends was developed, enabling the preferential sequencing of the 3' UTRs and identification of polyA sites in two colorectal cancer cell lines (HCT116 and SW480). PolyA variants with 3' UTR shorter than the reference transcript (RefSeq with the longest 3' UTR) were identified for more than 4000 genes, corresponding to 30% of expressed genes in these cells. About 60% of short polyA variants identified have lost at least one conserved miRNA target site. Specifically for these genes, there was a reduction on the ratio of long variants expression by short variants expression (LV/SV ratio) comparing to genes without miRNA target sites. Moreover, there was a significant difference of the LV/LS ratio between genes with target sites for non-expressed miRNAs and genes with target sites for expressed miRNAs. This reduction was even greater for those genes with short variants that lost target sites for highly expressed miRNAs when compared to genes with target sites for miRNAs with low expression. This innovative approach increased the capacity of identification and expression analysis of polyA variants and also confirmed the key role of miRNA expression over polyA variants expression...

Alternative polyadenylation site analysis of tumor-related genes based on 3'RACE in gastric cancer cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To analyze the alteration in alternative polyadenylation (APA) sites of tumor-related genes in gastric cancer cells.</p><p><b>METHODS</b>We 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.</p><p><b>RESULTS</b>We found new APA sites in the two tumor-related genes in gastric cancer cells to produce new mRNA isoforms with different 3'UTRs.</p><p><b>CONCLUSIONS</b>There 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.</p>

Conservation of cis-Regulatory Element Controlling Timely Translation in the 3'-UTR of Selected Mammalian Maternal Transcripts

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.

5' and 3' cis-Acting RNA Elements Required for RNA Replication of Porcine Reproductive and Respiratory Syndrome Virus

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.

Caracterização de um novo antígeno tumoral: CTSP-1 / Characterization of a new tumor antigen: CTSP-1

A necessidade de identificar novos antígenos tumorais que possam ser utilizados no tratamento e diagnóstico do câncer, tem levado ao desenvolvimento de técnicas cada vez mais eficientes na detecção dos mesmos. Antígenos de diferentes categorias foram identificados e caracterizados, sendo os antígenos cancer-testis (CT) e os antígenos de diferenciação (CD) os de maior importância clínica, dado seu restrito padrão de expressão. Utilizando uma estratégia de alinhamento de seqüências expressas no genoma humano, identificamos um novo transcrito localizado no cromossomo 21, denominado CTSP-1, que apresenta alta similaridade com o antígeno tumoral NY-BR-1... Além disso, avaliamos seu padrão de expressão em diferentes tecidos normais, linhagens celulares tumorais e amostras de tumores de pacientes... Através de immunoblotting foi possível a identificação de uma banda específica de 22kDa, correspondente ao peso esperado da proteína CTSP-1 em extrato total de testículo normal. Em seguida, através da imunohistoquímica, verificamos uma marcação preferencial nas espermatogônias e células de Leydig de testículo normal. Nos tecidos com amostras pareadas normal/tumor (mama e próstata), apenas as amostras tumorais foram fortemente marcadas. Posteriormente, a proteína CTSP-1 recombinante foi utilizada na investigação de anticorpos específicos em plasma de pacientes com câncer. Aproximadamente 150 amostras foram analisadas, das quais 20% apresentaram resposta imune humoral contra a proteína CTSP-1. Estes resultados revelam que o CTSP-1 é um novo antígeno tumoral da categoria dos CTs, com expressão restrita a tumor e testículo e com alta imunogenicidade em pacientes com câncer...(AU)

The need to identify new tumor antigens to be used in cancer treatment and diagnosis has lead to the development of efficient techniques for this purpose. Antigens from different categories have been identified and characterized and, among those, the cancer-testis (CT) and the cancer differentiation (CD) antigens are of the greatest clinicai interest dueto their restricted expression partem. Using alignments between expressed sequences and the Human Genome Sequence, we identified a new gene located on chromosome 21, named CTSP-1. This gene has a high similarity to the tumor antigen NY-BR-1, which encodes for a tissue specific transcription factor and is a potential target for cancer immunotherapy. In order to verify ifthe CTSP-1 gene is really a new tumor antigen, we performed its complete characterization. Using different techniques, we were able to obtain the complete sequence of the CTSP-1 gene and to identify different alternative polyadenilation and splicing forms. Moreover, we analyzed the CTSP-1 expression pattern in normal tissues, tumor cell lines and tumor samples. CTSP-1 showed a restricted expression pattern, being expressed only in testis among normal tissues, in different tumor celllines (9/22) and in different tumor types (7 4/178), which matches with the expression pattern of Cancer-Testis antigens. Afterwards, the recombinant CTSP-1 protein was expressed in a heterologous system and used for the generation of polyclonal antibody in mice. This antiboby was used in immunoblotting and immunohistochemistry experiments for the detection of CTSP-1 protein in normal testis and paired normal and tumor samples from breast and prostate. Using immunoblotting, a 22kDa specific band was identified in testis total protein extract, corresponding to the expected molecular weight of the CTSP-1 protein. Using immunohistochemistry, we verified the preferential staining of germ cells and Leydig cells in normal testis. Among tissues with paired normal/tumor samples, only tumor samples were strongly stained. The CTSP-1 recombinant protein was also used in the search for specific antibodies in plasma from cancer patients. Approximately 150 samples were analyzed, of which 20% showed a humoral immune response against the CTSP-1 protein. Taken together, these results confirm that the CTSP-1 gene is a new tumor antigen from the Cancer-Testis category, with restricted expression in testis and tumors and with high immunogenicity in cancer patients (AU)

Expression of Exon 1 and 6 of Indulin-like Growth Factor / 대한내분비학회지

Background: Goiter has been a common problem in the thyroid disease. The exact mechanism of goiter had not been clarified yet, but some goiters were increased with TSH(thyrotropin releasing hormone) dependent manner. TSH might be a major influencing factor for increasing size of goiter(goitrogen) and theres many cofactors those influenced to goiter size. One of the rnost prominent growth factor as a goitrogen is a IGF-I(insulin-like growth factor-I). IGF-I play a great role as a cofactor of goitrogen with TSH. This study, therefore, is aimed to investigate intracellular activation of IGF-I gene promoter in the surgical specimens of thyroid tumor. Methods: We used surgical specimen of various thyroid tissues from normal to malignant along its cell nature. Actually we used normal liver tissue as a IGF-I control tissue, normal thyroid, benign adenoma, and papillary thyroid cancer tissue with its malignat nature. We checked Mrna expression of whole IGF-I and IGF-I exon 6 by Northern blot method, and IGF-I, promoter 1 expression by RT-PCR-transcription method. Autoradiographied signals were analysed with densitometer. Results: We found whole IGF-I mRNAs were expressed with alternate splicing in exon 1, 2 and exon 4, 5 respectively. Striking events of IGF-I transcription were multiple tranascription initiatian in Pl and P2, and 3 sites for polyadenylation in exon 6. Four or more Mrna bands in Northern blot analysis of IGF-I(0.8, 1.4, 4.2, and 7.8kb) were noted. In low molecular weight IGF-I Mrna did not change their signal intensity with tissues, but exan 6(7.8kb) signals were significantly increased to its hepatic expression levels in malignant tissue. IGF-I, exon 1 expression by RT-PCR-T7 transcription was strikingly increased in thyroid cancer tissue, but exon 6 expression was not a great expession. Conclusion: One possible guess for this expression discrepancy of each exon may be originated from different Mrna degradation of each IGF-I signals. We need more preeise experiment for Mrna degradation speed of IGF-I.