Evidence that a consensus element found in naturally intronless mRNAs promotes mRNA export

We previously showed that mRNAs synthesized from three genes that naturally lack introns contain a portion of their coding sequence, known as a cytoplasmic accumulation region (CAR), which is essential for stable accumulation of the intronless mRNAs in the cytoplasm. The CAR in each mRNA is unexpectedly large, ranging in size from ∼160 to 285 nt. Here, we identified one or more copies of a 10-nt consensus sequence in each CAR. To determine whether this element (designated CAR-E) functions in cytoplasmic accumulation of intronless mRNA, we multimerized the most conserved CAR-E and inserted it upstream of β-globin cDNA, which is normally retained/degraded in the nucleus. Significantly, the tandem CAR-E, but not its antisense counterpart, rescued cytoplasmic accumulation of β-globin cDNA transcripts. Moreover, dinucleotide mutations in the CAR-E abolished this rescue. We show that the CAR-E, but not the mutant CAR-E, associates with components of the TREX mRNA export machinery, the Prp19 complex and U2AF2. Moreover, knockdown of these factors results in nuclear retention of the intronless mRNAs. Together, these data suggest that the CAR-E promotes export of intronless mRNA by sequence-dependent recruitment of the mRNA export machinery

Estrogen regulates Hippo signaling via GPER in breast cancer

The G protein–coupled estrogen receptor (GPER) mediates both the genomic and nongenomic effects of estrogen and has been implicated in breast cancer development. Here, we compared GPER expression in cancerous tissue and adjacent normal tissue in patients with invasive ductal carcinoma (IDC) of the breast and determined that GPER is highly upregulated in cancerous cells. Additionally, our studies revealed that GPER stimulation activates yes-associated protein 1 (YAP) and transcriptional coactivator with a PDZ-binding domain (TAZ), 2 homologous transcription coactivators and key effectors of the Hippo tumor suppressor pathway, via the Gαq-11, PLCβ/PKC, and Rho/ROCK signaling pathways. TAZ was required for GPER-induced gene transcription, breast cancer cell proliferation and migration, and tumor growth. Moreover, TAZ expression positively correlated with GPER expression in human IDC specimens. Together, our results suggest that the Hippo/YAP/TAZ pathway is a key downstream signaling branch of GPER and plays a critical role in breast tumorigenesis

Identification of splicing silencers and enhancers in sense Alus: a role for pseudoacceptors in splice site repression

Auxiliary splicing signals in introns play an important role in splice site selection, but these elements are poorly understood. We show that a subset of serine/arginine (SR)-rich proteins activate a cryptic 3' splice site in a sense Alu repeat located in intron 4 of the human LST1 gene. Utilization of this cryptic splice site is controlled by juxtaposed Alu-derived splicing silencers and enhancers between closely linked short tandem repeats TNFd and TNFe. Systematic mutagenesis of these elements showed that AG dinucleotides that were not preceded by purine residues were critical for repressing exon inclusion of a chimeric splicing reporter. Since the splice acceptor-like sequences are present in excess in exonic splicing silencers, these signals may contribute to inhibition of a large number of pseudosites in primate genomes

Functional analysis of genetic variants in putative low penetrate breast cancer genes

Paper I & II CDH1 germline mutations predispose individuals to diffuse gastric cancer, but its role in breast cancer is less clear. Somatic CDH1 mutations were reported to be frequent in lobular tumours (ILC), but they have not been found in ductal carcinomas (IDC). To define the role of CDH1 in breast cancer, we used denaturing high performance liquid chromatography to screen a series of breast cancer samples for mutations. Somatic mutations were detected in 4 of 83 IDC (5%) and 3 of 25 ILC (12%). No germline mutation was found in 19 familial breast cancer patients that showed loss of heterozygosity (LOH) at 16q24, in 12 cases from 10 families with breast, gastric and colon cancer and in 13 familial lobular breast tumours. Another somatic mutation was detected in one of the familial breast cancer patient with both ductal and lobular foci. Putative breast cancer risk conferred by the promoter polymorphism -161C-A of CDH1 or 1774G-A (Ala592Thr) was also analyzed in case-control studies. No significant difference in allelic frequency was found between the breast cancer patients and controls for either polymorphism. A novel promoter polymorphism was identified at position -152 with a similar frequency of the rare C allele in both breast cancer patients and controls. Transient transfection assay using constructs containing -16IC/-152C or -161A/-152T showed only a slight decrease of the transcription activity as compared to the wild type constructs carrying -161C/152T. We conclude that CDH1 is not a prominent lowpenetrance gene in breast cancer, but CDH1 mutations contribute to the progression of both lobular and ductal tumours. Paper III BACH1, a gene located in 17q22 and encoding a protein directly interacting with BRCA1, was suggested to be a candidate gene for breast cancer susceptibility. Using PCR-SSCP, we screened for germline BACH] mutations with 29 breast cancer families linked to 17q22 and additional 95 familial breast cancer cases, which were all without detectable BRCA112 mutations. No mutation was found. A C/T polymorphism at position 517 was detected, which leads to Arg173Cys substitution in the putative nuclear localization sequence. This alteration may contribute to the development of breast cancer, but BACH1 is not a major breast cancer gene. Paper IV ATM has been suggested to act as a tumour suppression gene that requires the inactivation of both alleles in the development of a malignancy. Two mutations designated as T7271G and IVS1O+6 T-G were reported to increase breast cancer risk in multiple-case families in a dominant negative manner. We evaluated the population frequency of these two mutations in Sweden and Czech populations using PCR-RFLP. The mutation T7271G was not detected, mutation IVS 10+6 T-G was found in 2 of 768 cases and I in 557 controls, giving the allelic frequency of 0. 1%. We also tested the hypothesis that ATM mutations would be enriched in breast cancer patients with LOH at 11q22-23 if ATM acted as TSG. Forty-two selected DNA samples from breast cancer cases were screened for mutations in ATM from exons 50 to 66 using PCR-SSCP, but no mutation was detected. Paper V (manuscript) LST1 is a gene located in the TNFalpha region and producing many isoforms with a possible role in immune response. In an attempt to understand the mechanism of the 3' splicing site selection in LST1, we found that removal of a nearby AG dinucleotide repeat TNFd led to a serine-arginine protein-dependent activation of a cryptic 3' splice site. The highest yield of the novel isoform named LST1/n was induced by ASF and SRp40. This effect was dose-dependent, it was also dependent on the length of TNFd. Deletion of RNA recognition motif 2 (RRM2) of ASF/SF2 abolished the usage of the cryptic splice site, indicating that the activation is mediated by RNA binding. In addition, we showed that hnRNP Al but not hnRNP K could effectively antagonize SR-protein induced activation. Replacement of TNFd with AC or AT repeat indicated a AG-AC>AT repressing hierarchy on the SR protein induced activation. Further removal of a putative splicing silencer A3 alleviated the requirement for exogenous SR proteins in LST/n activation. We also showed that lack of this event in the wild type construct was not due to the differential efficiency in RTPCR for TNFd+ or TNFd- isoform. Insertion of TNFd in a heterologous context showed no evidence of splicing inhibition in in vitro splicing analysis. Insertion of A3 into a middle exon of a heterologousACE Alu + 136/3'ss minigene led to almost full exon skipping, suggesting that A3 contains splicing inhibitory sequences. All these data are consistent with the existence of multiple mechanisms in the repression of pseudo splice sites and with the concept that dinucleotide repeats could act as splicing regulatory elements in the vicinity of splice sites

Exonization of AluYa5 in the human ACE gene requires mutations in both 3' and 5' splice sites and is facilitated by a conserved splicing enhancer

Ancient Alu elements have been shown to be included in mature transcripts by point mutations that improve their 5' or 3' splice sites. We have examined requirements for exonization of a younger, disease-associated AluYa5 in intron 16 of the human ACE gene. A single G>C transversion in position –3 of the new Alu exon was insufficient for Alu exonization and a significant inclusion in mRNA was only observed when improving several potential splice donor sites in the presence of 3' CAG. Since complete Alu exonization was not achieved by optimizing traditional splicing signals, including the branch site, we tested whether auxiliary elements in AluYa5 were required for constitutive inclusion. Exonization was promoted by a SELEX-predicted heptamer in Alu consensus sequence 222–228 and point mutations in highly conserved nucleotides of this heptamer decreased Alu inclusion. In addition, we show that Alu exonization was facilitated by a subset of serine/arginine-rich (SR) proteins through activation of the optimized 3' splice site. Finally, the haplotype- and allele-specific ACE minigenes generated similar splicing patterns in both ACE-expressing and non-expressing cells, suggesting that previously reported allelic association with plasma ACE activity and cardiovascular disease is not attributable to differential splicing of introns 16 and 17

Efficient keyword search on uncertain graph data

As a popular search mechanism, keyword search has been applied to retrieve useful data in documents, texts, graphs, and even relational databases. However, so far, there is no work on keyword search over uncertain graph data even though the uncertain graphs have been widely used in many real applications, such as modeling road networks, influential detection in social networks, and data analysis on PPI networks. Therefore, in this paper, we study the problem of top-k keyword search over uncertain graph data. Following the similar answer definition for keyword search over deterministic graphs, we consider a subtree in the uncertain graph as an answer to a keyword query if 1) it contains all the keywords; 2) it has a high score (defined by users or applications) based on keyword matching; and 3) it has low uncertainty. Keyword search over deterministic graphs is already a hard problem as stated in [1], [2], [3]. Due to the existence of uncertainty, keyword search over uncertain graphs is much harder. Therefore, to improve the search efficiency, we employ a filtering-and-verification strategy based on a probabilistic keyword index, PKIndex. For each keyword, we offline compute path-based top-k probabilities, and attach these values to PKIndex in an optimal, compressed way. In the filtering phase, we perform existence, path-based and tree-based probabilistic pruning phases, which filter out most false subtrees. In the verification, we propose a sampling algorithm to verify the candidates. Extensive experimental results demonstrate the effectiveness of the proposed algorithms. © 1989-2012 IEEE