Shortening of 3′UTRs Correlates with Poor Prognosis in Breast and Lung Cancer

A major part of the post-transcriptional regulation of gene expression is affected by trans-acting elements, such as microRNAs, binding the 3′ untraslated region (UTR) of their target mRNAs. Proliferating cells partly escape this type of negative regulation by expressing shorter 3′ UTRs, depleted of microRNA binding sites, compared to non-proliferating cells. Using large-scale gene expression datasets, we show that a similar phenomenon takes place in breast and lung cancer: tumors expressing shorter 3′ UTRs tend to be more aggressive and to result in shorter patient survival. Moreover, we show that a gene expression signature based only on the expression ratio of alternative 3′ UTRs is a strong predictor of survival in both tumors. Genes undergoing 3′UTR shortening in aggressive tumors of the two tissues significantly overlap, and several of them are known to be involved in tumor progression. However the pattern of 3′ UTR shortening in aggressive tumors in vivo is clearly distinct from analogous patterns involved in proliferation and transformation

Alternative Splicing in the Differentiation of Human Embryonic Stem Cells into Cardiac Precursors

The role of alternative splicing in self-renewal, pluripotency and tissue lineage specification of human embryonic stem cells (hESCs) is largely unknown. To better define these regulatory cues, we modified the H9 hESC line to allow selection of pluripotent hESCs by neomycin resistance and cardiac progenitors by puromycin resistance. Exon-level microarray expression data from undifferentiated hESCs and cardiac and neural precursors were used to identify splice isoforms with cardiac-restricted or common cardiac/neural differentiation expression patterns. Splice events for these groups corresponded to the pathways of cytoskeletal remodeling, RNA splicing, muscle specification, and cell cycle checkpoint control as well as genes with serine/threonine kinase and helicase activity. Using a new program named AltAnalyze (http://www.AltAnalyze.org), we identified novel changes in protein domain and microRNA binding site architecture that were predicted to affect protein function and expression. These included an enrichment of splice isoforms that oppose cell-cycle arrest in hESCs and that promote calcium signaling and cardiac development in cardiac precursors. By combining genome-wide predictions of alternative splicing with new functional annotations, our data suggest potential mechanisms that may influence lineage commitment and hESC maintenance at the level of specific splice isoforms and microRNA regulation

Mammalian microRNAs: a small world for fine-tuning gene expression

The basis of eukaryotic complexity is an intricate genetic architecture where parallel systems are involved in tuning gene expression, via RNA-DNA, RNA-RNA, RNA-protein, and DNA-protein interactions. In higher organisms, about 97% of the transcriptional output is represented by noncoding RNA (ncRNA) encompassing not only rRNA, tRNA, introns, 5′ and 3′ untranslated regions, transposable elements, and intergenic regions, but also a large, rapidly emerging family named microRNAs. MicroRNAs are short 20-22-nucleotide RNA molecules that have been shown to regulate the expression of other genes in a variety of eukaryotic systems. MicroRNAs are formed from larger transcripts that fold to produce hairpin structures and serve as substrates for the cytoplasmic Dicer, a member of the RNase III enzyme family. A recent analysis of the genomic location of human microRNA genes suggested that 50% of microRNA genes are located in cancer-associated genomic regions or in fragile sites. This review focuses on the possible implications of microRNAs in post-transcriptional gene regulation in mammalian diseases, with particular focus on cancer. We argue that developing mouse models for deleted and/or overexpressed microRNAs will be of invaluable interest to decipher the regulatory networks where microRNAs are involved

Identification and functional analysis of novel genes expressed in the Anterior Visceral Endoderm

During early vertebrate development, the correct establishment of the body axes is critical. The anterior pole of the mouse embryo is established when Distal Visceral Endoderm (DVE) cells migrate to form the Anterior Visceral Endoderm (AVE). Symmetrical expression of Lefty1, Cer1 and Dkk1 determines the direction of DVE migration and the future anterior side. In addition to the establishment of the Anterior-Posterior axis, the AVE has also been implicated in anterior neural specification. To better understand the role of the AVE in these processes, we have performed a differential screening using Affymetrix GeneChip technology with AVE cells isolated from cer1P-EGFP transgenic mouse embryos. We found 175 genes which were upregulated in the AVE and 36 genes in the Proximal-posterior sample. Using DAVID software, we characterized the AVE cell population regarding cellular component, molecular function and biological processes. Among the genes that were found to be upregulated in the AVE, several novel genes were identified. Four of these transcripts displaying high-fold change in the AVE were further characterized by in situ hybridization in early stages of development in order to validate the screening. From those four selected genes, one, denominated Adtk1, was chosen to be functionally characterized by targeted inactivation in ES cells. Adtk1 encodes for a serine/threonine kinase. Adtk1 null mutants are smaller and present short limbs due to decreased mineralization, suggesting a potential role in chondrogenesis during limb development. Taken together, these data point to the importance of reporting novel genes present in the AVE.F.C.T.; IGC/FCG; IBB/CBME, L

Genetic studies of familial myeloproliferative disorders

Hereditary thrombocythemia (HT) is an autosomal dominant disorder with clinical features resembling sporadic essential thrombocythemia. HT families share similar clinical symptoms caused by heterogeneous genetic alterations. Inherited germ-line mutations in the thrombopoietin (TPO) gene and its receptor MPL have been found causing thrombocytosis in a number of HT families. Five reported mutations in the thrombopoietin gene are all located in the 5 prime untranslated region (5’UTR) and cause overproduction of Tpo protein by the same mechanism: increased translation efficiency for the mutant mRNAs. One mutation identified in the MPL gene is located at the transmembrane domain and results in a hyperactive receptor, thereby leading to thrombocytosis. All these germ-line mutations have not been found in sporadic patients and are only responsible for the etiology of some HT families, indicating that the occurrence of these germ-line mutations is a rare event. The disease-causing genes for many HT families remain unknown. Identifying genetic lesions in these families will increase our knowledge of the physiology of thrombopoiesis and some of these unknown genetic components may contribute to the pathogenesis in sporadic MPD patients. In the first part of the project for genetic studies of HT families, the TPO and MPL genes were analyzed by genomic DNA exon sequencing and linkage analysis. A splice donor mutation in the TPO gene was identified in a Polish family. This mutation was previously identified in a Dutch family and the reoccurrence of this rare mutation has not been reported to date. In order to determine whether the 6 mutation mutation in these two families arose de novo or from a founder effect, haplotype analysis was performed to examine polymorphic DNA sequences in the vicinity of the mutation using microsatellites and single nucleotide polymorphism (SNP) in these two families. Six microsatellite markers on the affected allele showed different sizes in PCR products and 3 SNPs close to the mutation differed in their sequences between the two families. We therefore concluded that the mutation in these two families occurred de novo. The previously reported MPL mutation at the transmembrane domain of MPL protein was identified in one of the HT families studied here. Recently, 5 additional HT families were found carrying this mutation. We conducted haplotype analysis using microsatellite markers in the MPL gene locus for the 6 HT families. Four microsatellite markers surrounding the MPL mutation showed identical sizes in the PCR products on the affected allele, suggesting that the MPL mutation occurred from a single founder event. This may explain the high frequency of this mutation in HT families. In a large US family with HT, where the TPO and MPL genes were excluded as disease causes, genome-wide linkage analysis was performed aiming to identify novel genetic component for the thrombocytosis phenotype. Two genetic regions with significant logarithm of odds (LOD) score values have been located using microsatellites and SNP chip arrays. Candidate gene sequencing revealed one novel polymorphism in the gelsolin gene, which encodes an actin-binding protein abundant in platelets. Gelsolin has multiple biological functions in addition to cytoskeletal actin modulation. Functional studies in cell proliferation assays and mouse bone marrow transplantation did not validate this polymorphism as an active disease causing mutation. Further studies on this polymorphism in platelet biogenesis are planned for the future. In addition, sequencing of all the candidate genes in the segregating regions is in progress. In a second project, genome-wide linkage analyses were performed using microsatellites and SNP chip arrays in a family with secondary polycythemia inherited in an autosomal recessive mode. Both parametric and nonparametric linkage analysis were conducted for this family. Five genetic regions were found linked to the disease phenotype. A few candidate genes were sequenced and studied, however no genetic variation was found so far. Additionally, no mutations were found in several genes involved in erythropoiesis and oxygen sensing pathway. Burst forming units-Erythroid cultures in hypoxia condition showed high expression of the EPO gene in 3 out of 4 affected family members, suggesting a potential unknown defect in the oxygen-sensing pathway

Mammalian Cis-Acting RNA Sequence Elements

Cis-acting regulatory sequence elements are sequences contained in the 3′ and 5′ untranslated region, introns, or coding regions of precursor RNAs and mature mRNAs that are selectively recognized by a complementary set of one or more trans-acting factors to regulate posttranscriptional gene expression. This chapter focuses on mammalian cis-acting regulatory elements that had been recently discovered in different regions: pre-processed and mature. The chapter begins with an overview of two large networks of mRNAs that contain conserved AU-rich elements (AREs) or GU-rich elements (GREs), and their role in mammalian cell physiology. Other, less conserved, cis-acting elements and their functional role in different steps of RNA maturation and metabolism will be discussed. The molecular characteristics of pathological cis-acting sequences that rose from gene mutations or transcriptional aberrations are briefly outlined, with the proposed approach to restore normal gene expression. Concise models of the function of posttranscriptional regulatory networks within different cellular compartments conclude this chapter

The role of post-transcriptional regulation in chemokine gene expression in inflammation and allergy.

The aim of this review is to discuss recent advances in the understanding of the regulation of chemokine expression occurring during chronic inflammatory conditions, such as allergic diseases. The focus will be on current data, which suggest that post-transcriptional regulation plays a larger role in chemokine gene regulation than previously recognised. In particular, a growing body of data indicates that mechanisms controlling mRNA stability may be relevant in determining, or maintaining, the increased levels of chemokine gene expression in this context. Such regulatory pathways may be important targets of novel anti-inflammatory strategies

Reprogramming of 3′ Untranslated Regions of mRNAs by Alternative Polyadenylation in Generation of Pluripotent Stem Cells from Different Cell Types

The 3' untranslated regions (3'UTRs) of mRNAs contain cis elements involved in post-transcriptional regulation of gene expression. Over half of all mammalian genes contain multiple polyadenylation sites that lead to different 3'UTRs for a gene. Studies have shown that the alternative polyadenylation (APA) pattern varies across tissues, and is dynamically regulated in proliferating or differentiating cells. Generation of induced pluripotent stem (iPS) cells, in which differentiated cells are reprogrammed to an embryonic stem (ES) cell-like state, has been intensively studied in recent years. However, it is not known how 3'UTRs are regulated during cell reprogramming.Using a computational method that robustly examines APA across DNA microarray data sets, we analyzed 3'UTR dynamics in generation of iPS cells from different cell types. We found that 3'UTRs shorten during reprogramming of somatic cells, the extent of which depends on the type of source cell. By contrast, reprogramming of spermatogonial cells involves 3'UTR lengthening. The alternative polyadenylation sites that are highly responsive to change of cell state in generation of iPS cells are also highly regulated during embryonic development in opposite directions. Compared with other sites, they are more conserved, can lead to longer alternative 3'UTRs, and are associated with more cis elements for polyadenylation. Consistently, reprogramming of somatic cells and germ cells involves significant upregulation and downregulation, respectively, of mRNAs encoding polyadenylation factors, and RNA processing is one of the most significantly regulated biological processes during cell reprogramming. Furthermore, genes containing target sites of ES cell-specific microRNAs (miRNAs) in different portions of 3'UTR are distinctively regulated during cell reprogramming, suggesting impact of APA on miRNA targeting.Taken together, these findings indicate that reprogramming of 3'UTRs by APA, which result from regulation of both general polyadenylation activity and cell type-specific factors and can reset post-transcriptional gene regulatory programs in the cell, is an integral part of iPS cell generation, and the APA pattern can be a good biomarker for cell type and state, useful for sample classification. The results also suggest that perturbation of the mRNA polyadenylation machinery or RNA processing activity may facilitate generation of iPS cells