Activation of signal transducers and activators of transcription (STAT) proteins may be critical to their oncogenic functions as demonstrated by the development of B-cell lymphoma/leukemia in transgenic (TG) mice overexpressing a constitutively activated form of Stat5b. However, low incidence of CD8+ T cell lymphoma was observed in B6 transgenic mice overexpressing a wild-type Stat5b (B6.Stat5bTg) despite of undetectable Stat5b phosphorylation and the rate of lymphomagenesis was markedly enhanced by immunization or the introduction of TCR transgenes . Here, we report that the wild-type Stat5b transgene leads to the acceleration and high incidence (74%) of CD8+ T cell lymphoblastic lymphomas in the non-obese-diabetic (NOD) background. In contrast to the B6.Stat5bTg mice, Stat5b in transgenic NOD (NOD.Stat5bTg) mice is selectively and progressively phosphorylated in CD8+ thymocytes. Stat5 phosphorylation also leads to up-regulation of many genes putatively relevant to tumorigenesis. Treatment of NOD.Stat5bTg mice with cancer chemopreventive agents Apigenin and Xanthohumol efficiently blocked lymphomagenesis through reduction of Stat5 phosphorylation and genes up-regulated in the NOD.Stat5bTg mice. These results suggest that NOD genetic background is critical to the Stat5b-mediated lymphomagenesis through regulation of Stat5 hyperactivation. NOD.Stat5bTg mouse is an excellent model for studying the molecular mechanisms underlying lymphomagenesis and testing novel chemoprevention strategies.
There is no consensus regarding the management of ovarian cancer patients, who have shown complete clinical response (CCR) to primary therapy and have rising cancer antigen CA-125 levels but have no symptoms of recurrent disease. The present study aims to determine whether follow-up CA-125 levels can be used to identify the need for imaging studies and secondary cytoreductive surgery (CRS).
We identified 410 ovarian cancer patients treated at The University of Texas MD Anderson Cancer Center between 1984 and 2011. These patients had shown CCR to primary therapy. Follow-up was conducted based on the surveillance protocol of the MD Anderson Cancer Center. We used the Cox proportional hazards model and log-rank test to assess the associations between the follow-up CA-125 levels and secondary CRS and survival duration.
The CA-125 level of 1.68 × nadir was defined as the indicator of recurrent disease (p < 0.001). The specificity and sensitivity of this criterion were 82.9% and 85.6%, respectively, and the median lead-time of the CA-125 biochemical progression prior to clinically-defined relapse was 31 days (ranging from 1 to 391 days). The median number of the negative imaging studies for the clinical relapse findings in patients with a CA-125 level of < 1.68 × nadir was 3 (ranging from 0 to 24 times). The increase of CA-125 level at relapse was an independent predictor of overall and progression free survival in patients who had shown CCR to primary therapy (p = 0.04 and 0.02 respectively). The overall and progression free survival durations in patients with a CA-125 level ≤ 1.68 × nadir at relapse (69.4 and 13.8 months) were longer than those with a CA-125 level > 1.68 × nadir at relapse (55.7 and 10.4 months; p = 0.04 and 0.01, respectively). The overall and progression free survival duration of patients with asymptomatic relapse and underwent a secondary CRS was longer than that of patients with symptomatic relapse (p = 0.02 and 0.04 respectively).
The increase of serum CA-125 levels is an early warning of clinical relapse in ovarian cancer. Using CA-125 levels in guiding the treatment of patients with asymptomatic recurrent ovarian cancer, who have shown CCR to primary therapy, can facilitate optimal secondary CRS and extend the survival duration of the patients.
Epithelial ovarian cancer; CA-125; Tumor marker; Clinical relapse; Cytoreductive surgery
The Wnt signaling pathway plays an important role not only in embryonic development but also in the maintenance and differentiation of the stem cells in adulthood. In particular, Wnt signaling has been shown as an important regulatory pathway in the osteogenic differentiation of mesenchymal stem cells. Induction of the Wnt signaling pathway promotes bone formation while inactivation of the pathway leads to osteopenic states. Our current understanding of Wnt signaling in osteogenesis elucidates the molecular mechanisms of classic osteogenic pathologies. Activating and inactivating aberrations of the canonical Wnt signaling pathway in osteogenesis results in sclerosteosis and osteoporosis respectively. Recent studies have sought to target the Wnt signaling pathway to treat osteogenic disorders. Potential therapeutic approaches attempt to stimulate the Wnt signaling pathway by upregulating the intracellular mediators of the Wnt signaling cascade and inhibiting the endogenous antagonists of the pathway. Antibodies against endogenous antagonists, such as sclerostin and dickkopf-1, have demonstrated promising results in promoting bone formation and fracture healing. Lithium, an inhibitor of glycogen synthase kinase 3β, has also been reported to stimulate osteogenesis by stabilizing β catenin. Although manipulating the Wnt signaling pathway has abundant therapeutic potential, it requires cautious approach due to risks of tumorigenesis. The present review discusses the role of the Wnt signaling pathway in osteogenesis and examines its targeted therapeutic potential.
Wnt signaling; bone formation; osteoporosis; fracture healing; bone tumors
The members of the Aurora kinase family play critical roles in the regulation of the cell cycle and mitotic spindle assembly and have been intensively investigated as potential targets for a new class of anti-cancer drugs. We describe a new highly potent and selective class of Aurora kinase inhibitors discovered using a phenotypic cellular screen. Optimized inhibitors display many of the hallmarks of Aurora inhibition including endoreduplication, polyploidy, and loss of cell viability in cancer cells. Structure-activity relationships with respect to kinome-wide selectivity and guided by an Aurora B co-crystal structure resulted in the identification of key selectivity determinants and discovery of a sub-series with selectivity towards Aurora A. A direct comparison of biochemical and cellular profile with respect to published Aurora inhibitors including VX-680, AZD1152, MLN8054, and a pyrimidine-based compound from Genentech demonstrates that compounds 1 and 3 will become valuable additional pharmacological probes of Aurora dependent functions.
In absence of their natural ligand 11-cis retinal, cone opsin GPCRs fail to traffic normally, a condition associated with photoreceptor degeneration and blindness. We created a mouse with a point mutation (F81Y) in cone S-opsin. As expected, cones with this knockin mutation respond to light with maximal sensitivity red-shifted from 360 nm to 420 nm, consistent with an altered interaction between the apoprotein and ligand, 11-cis retinal. However, cones expressing F81Y S-opsin showed an ~3-fold reduced absolute sensitivity that was associated with a corresponding reduction in S-opsin protein expression. The reduced S-opsin expression did not arise from decreased S-opsin mRNA or cone degeneration, but rather from enhanced ER-associated degradation (ERAD) of the nascent protein. Exogenously increased 11-cis retinal restored F81Y S-opsin protein expression to normal levels, suggesting that ligand binding in the ER facilitates proper folding. Immunohistochemistry and electron microscopy of normal retinas showed that Mueller cells, which synthesize a precursor of 11-cis retinal, are closely adjoined to the cone ER, so could deliver the ligand to the site of opsin synthesis. Together, these results suggest that the binding of 11-cis retinal in the ER is important for normal folding during cone opsin biosynthesis.
ER quality control; ERAD; retinal degeneration; photoreceptor; retinoid
The development of new effective therapeutic agents with minimal side effects for prostate cancer treatment is much needed. Indirubin, an active molecule identified in the traditional Chinese herbal medicine – Qing Dai (Indigo Naturalis), has been used to treat leukemia for decades. However, the anti-cancer properties of Natura-alpha, an indirubin derivative, are not well studied in solid tumors, particularly in prostate cancer.
Human prostate cancer cell lines were treated with or without Natura-alpha followed by cell growth and invasion assays measured. The anti-tumor effects of Natura-alpha were examined in nude mice tumor xenograft models, as well as in a patient with advanced hormone refractory metastatic prostate cancer. Signal network proteins targeted by Natura-alpha were analyzed using Proteomic Pathway Array Analysis (PPAA) on xenografts.
Natura-alpha inhibited the growth of both androgen-dependent (LNCaP), and androgen-independent (LNCaP-AI, PC-3, and DU145) prostate cancer cells with IC50 between 4 to 10 Μm, also inhibits invasion of androgen-independent prostate cancer cells. Its anti-tumor effects were further evident in vivo tumor reduction in androgen-dependent and -independent nude mice tumor xenograft models as well as reduced tumor volume in the patient with hormone refractory metastatic prostate cancer. PPAA revealed that anti-proliferative and anti-invasive activities of Natura-alpha on prostate cancer might primarily be through its down-regulation of Forkhead box M1 (FOXM1) protein. Forced over-expression of FOXM1 largely reversed the inhibition by Natura-alpha.
Natura-alpha could serve as a novel and effective therapeutic agent for treatment of both hormone sensitive and hormone refractory prostate cancer with minimal side effects.
Superficial spreading melanoma (SSM) and nodular melanoma (NM) are believed to represent sequential phases of linear progression from radial to vertical growth. Several lines of clinical, pathological and epidemiologic evidence suggest, however, that SSM and NM might be the result of independent pathways of tumor development. We utilized an integrative genomic approach that combines single nucleotide polymorphism array (SNP 6.0, Affymetrix) with gene expression array (U133A 2.0, Affymetrix) to examine molecular differences between SSM and NM. Pathway analysis of the most differentially expressed genes between SSM and NM (N=114) revealed significant differences related to metabolic processes. We identified 8 genes (DIS3, FGFR1OP, G3BP2, GALNT7, MTAP, SEC23IP, USO1, ZNF668) in which NM/SSM-specific copy number alterations correlated with differential gene expression (P<0.05, Spearman’s rank). SSM-specific genomic deletions in G3BP2, MTAP, and SEC23IP were independently verified in two external data sets. Forced overexpression of metabolism-related gene methylthioadenosine phosphorylase (MTAP) in SSM resulted in reduced cell growth. The differential expression of another metabolic related gene, aldehyde dehydrogenase 7A1 (ALDH7A1), was validated at the protein level using tissue microarrays of human melanoma. In addition, we show that the decreased ALDH7A1 expression in SSM may be the result of epigenetic modifications. Our data reveal recurrent genomic deletions in SSM not present in NM, which challenge the linear model of melanoma progression. Furthermore, our data suggest a role for altered regulation of metabolism-related genes as a possible cause of the different clinical behavior of SSM and NM.
melanoma; nodular; genomics; SNP array; DNA copy number
The mTOR mediated PI3K/AKT/mTOR signal transduction pathway has been demonstrated to play a key role in a broad spectrum of cancers. Starting from the mTOR selective inhibitor 1 (Torin1), a focused medicinal chemistry effort led to the discovery of an improved mTOR inhibitor 3 (Torin2), which possesses an EC50 of 0.25 nM for inhibiting cellular mTOR activity. Compound 3 exhibited 800-fold selectivity over PI3K (EC50: 200 nM) and over 100-fold binding selectivity relative to 440 other protein kinases. Compound 3 has significantly improved bioavailability (54%), metabolic stability and plasma exposure relative to compound 1.
Mouse cone photoreceptors, like those of most mammals including humans, express cone opsins derived from two ancient families: S-opsin (gene Opn1sw) and M-opsin (gene Opn1mw). Most C57BI/6 mouse cones co-express both opsins, but in dorso-ventral counter-gradients, with M-opsin dominant in the dorsal retina and S-opsin in the ventral retina, and S-opsin 4-fold greater overall. We created a mouse lacking S-opsin expression by the insertion of a Neomycin selection cassette between the third and fourth exons of the Opn1sw gene (Opn1swNeo/Neo). In strong contrast to published results characterizing mice lacking rhodopsin (Rho−/−) in which retinal rods undergo cell death by 2.5 months, cones of the Opn1swNeo/Neo mouse remain viable for at least 1.5 yrs, even though many ventral cones do not form outer segments, as revealed by high resolution immunohistochemistry and electron microscopy. Suction pipette recordings revealed that functional ventral cones of the Opn1swNeo/Neo mouse not only phototransduce light with normal kinetics, but are more sensitive to mid-wavelength light than their WT counterparts. Quantitative Western blot analysis revealed the basis of the heightened sensitivity to be increased M-opsin expression. Because S- and M-opsin transcripts must compete for the same translational machinery in cones where they are co-expressed, elimination of S-opsin mRNA in ventral Opn1swNeo/Neo cones likely increases M-opsin expression by relieving competition for translational machinery, revealing an important consequence of eliminating a dominant transcript. Overall, our results reveal a striking capacity for cone photoreceptors to function with much reduced opsin expression, and to remain viable in the absence of an outer segment.
opsin; cone survival; phototransduction; color vision
Mesenchymal stem cells (MSCs) are multipotent cells which reside in many tissues and can give rise to multiple lineages including bone, cartilage and adipose. Although MSCs have attracted significant attention for basic and translational research, primary MSCs have limited life span in culture which hampers MSCs' broader applications. Here, we investigate if mouse mesenchymal progenitors can be conditionally immortalized with SV40 large T antigen and maintain long-term cell proliferation without compromising their multipotency. Using the system which expresses SV40 large T antigen flanked with Cre/loxP sites, we demonstrate that mouse embryonic fibroblasts (MEFs) can be efficiently immortalized by SV40 large T antigen. The conditionally immortalized MEFs (iMEFs) exhibit an enhanced proliferative activity and maintain long-term cell proliferation, which can be reversed by Cre recombinase. The iMEFs express most MSC markers and retain multipotency as they can differentiate into osteogenic, chondrogenic and adipogenic lineages under appropriate differentiation conditions in vitro and in vivo. The removal of SV40 large T reduces the differentiation potential of iMEFs possibly due to the decreased progenitor expansion. Furthermore, the iMEFs are apparently not tumorigenic when they are subcutaneously injected into athymic nude mice. Thus, the conditionally immortalized iMEFs not only maintain long-term cell proliferation but also retain the ability to differentiate into multiple lineages. Our results suggest that the reversible immortalization strategy using SV40 large T antigen may be an efficient and safe approach to establishing long-term cell culture of primary mesenchymal progenitors for basic and translational research, as well as for potential clinical applications.
The multi-subunit Sin3 co-repressor complex regulates gene transcription through deacetylation of nucleosomes. However, the full range of Sin3 activities and targets is not well understood. Here, we have investigated genome-wide binding of mouse Sin3 and RBP2 as well as histone modifications and nucleosome positioning as a function of myogenic differentiation. Remarkably, we find that Sin3 complexes spread immediately downstream of the transcription start site on repressed and transcribed genes during differentiation. We show that RBP2 is part of a Sin3 complex, and on a subset of E2F4 target genes, the coordinated activity of Sin3 and RBP2 leads to deacetylation, demethylation, and repositioning of nucleosomes. Our work provides evidence for coordinated binding of Sin3, chromatin modifications, and chromatin remodeling within discrete regulatory regions, suggesting a model in which spreading of Sin3 binding is ultimately linked to permanent gene silencing on a subset of E2F4 target genes.
To evaluate the antioxidant and free-radical scavenging activities of triethylchebulate (TCL), an aglycone isolated from the fruit of Terminalia chebula Retz.
Materials and Methods:
Microsomes, mitochondria and red blood cells (RBCs) were isolated from rat liver. The antioxidant capacities were evaluated by determining the inhibitory effects of TCL on lipid peroxidation, hydrogen peroxide (H2O2)-induced RBCs hemolysis and RBCs autoxidative hemolysis. The free-radical scavenging activities were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) method and 2´,7´-dichlorodihydrofluorescin diacetate (DCFH2-DA) assay.
TCL significantly inhibited FeSO4/Cys-induced microsomes lipid peroxidation and protected both H2O2--induced RBCs hemolysis and RBCs auto-hemolysis in a dose-dependent manner. Furthermore, TCL demonstrated potent DPPH free-radical scavenging ability with IC50 at 2.4×10-5 M. In addition, TCL also moderately suppressed azide-induced mitochondria ROS formation.
These results demonstrated that TCL was a strong antioxidant and free-radical scavenger, which might contribute to the anti-oxidative ability of Terminalia chebula Retz.
Anti-oxidant; hemolysis; lipid peroxidation; reactive oxygen species; Terminalia chebula Retz; triethylchebulate
Engineered nanoparticles with theranostic functions have attracted a lot of attention for their potential role in the dawning era of personalized medicine. Iron oxide nanoparticles (IONPs), with their advantages of being non-toxic, biodegradable and inexpensive, are candidate platforms for the build-up of theranostic nanostructures; however, progress in using them has been limited largely due to inefficient drug loading and delivery. In the current study, we utilized dopamine to modify the surface of IONPs, yielding nanoconjugates that can be easily encapsulated into human serum albumin (HSA) matrices (clinically utilized drug carriers). This nanosystem is well-suited for dual encapsulation of IONPs and drug molecules, because the encapsulation is achieved in a way that is similar to common drug loading. To assess the biophysical characteristics of this novel nanosystem, the HSA coated IONPs (HSA-IONPs) were dually labeled with 64Cu-DOTA and Cy5.5, and tested in a subcutaneous U87MG xenograft mouse model. In vivo positron emission tomography (PET)/near-infrared fluorescence (NIRF)/magnetic resonance imaging (MRI) tri-modality imaging, and ex vivo analyses and histological examinations were carefully conducted to investigate the in vivo behavior of the nanostructures. With the compact HSA coating, the HSA-IONPs manifested a prolonged circulation half-life; more impressively, they showed massive accumulation in lesions, high extravasation rate, and low uptake of the particles by macrophages at the tumor area.
iron oxide nanoparticle (IONP); magnetic resonance imaging (MRI); positron emission tomography (PET); near-infrared fluorescence (NIRF) imaging; enhanced permeability and retention (EPR) effect
The c-Jun N-terminal kinase (JNK) mediates stress-induced apoptosis and the cytotoxic effect of anticancer therapies. Paradoxically, recent clinical studies indicate that elevated JNK activity in human breast cancer is associated with poor prognosis. Here we show that overexpression of a constitutively active JNK in human breast cancer cells did not cause apoptosis, but actually induced cell migration and invasion, a morphological change associated with epithelial-mesenchymal transition (EMT), expression of mesenchymal-specific markers vimentin and fibronectin, and activity of AP-1 transcription factors. Supporting this observation, mouse mammary tumor cells that have undergone EMT showed upregulated JNK activity, and the EMT was reversed by JNK inhibition. Sustained JNK activity enhanced insulin receptor substrate-2-mediated ERK activation, which in turn increased c-Fos expression and AP-1 activity. In addition, hyperactive JNK attenuated the apoptosis of breast cancer cells treated by the chemotherapy drug paclitaxel, which is in contrast to the requirement for inducible JNK activity in response to cytotoxic chemotherapy. Blockade of ERK activity diminished hyperactive JNK-induced cell invasion and survival. Our data suggest that the role of JNK changes when its activity is elevated persistently above the basal levels associated with cell apoptosis, and that JNK activation may serve as a marker of breast cancer progression and resistance to cytotoxic drugs.
AP-1; breast cancer; epithelial-mesenchymal transition; ERK; IRS-2; JNK
Perchlorotrityl radicals, monosubstituted with a fluorophore using an amide linker of varying chain length, were synthesized and characterized. Electron paramagnetic resonance (EPR) spectroscopic study indicated free-electron coupling with the aromatic hydrogen nuclei and long-range coupling with the methylene hydrogens of the linker group. Reactivity of the fluorophore-conjugated trityls with superoxide radical anion showed quenching of EPR signal and enhancement of fluorescence emission spectrum. This work presents the first example of a perchlorotrityl-fluorophore conjugate that can potentially be employed as a dual probe for the detection of superoxide under oxidative stress-mediated conditions in biological systems.
Cancer cells have an efficient antioxidant system to counteract their increased generation of ROS. However, whether this ability to survive high levels of ROS has an important role in the growth and metastasis of tumors is not well understood. Here, we demonstrate that the redox protein thioredoxin-like 2 (TXNL2) regulates the growth and metastasis of human breast cancer cells through a redox signaling mechanism. TXNL2 was found to be overexpressed in human cancers, including breast cancers. Knockdown of TXNL2 in human breast cancer cell lines increased ROS levels and reduced NF-κB activity, resulting in inhibition of in vitro proliferation, survival, and invasion. In addition, TXNL2 knockdown inhibited tumorigenesis and metastasis of these cells upon transplantation into immunodeficient mice. Furthermore, analysis of primary breast cancer samples demonstrated that enhanced TXNL2 expression correlated with metastasis to the lung and brain and with decreased overall patient survival. Our studies provided insight into redox-based mechanisms underlying tumor growth and metastasis and suggest that TXNL2 could be a target for treatment of breast cancer.
The mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase and the founding member of a signaling pathway that regulates many fundamental features of cell growth and division. In cells, mTOR acts as the catalytic subunit of two functionally distinct complexes, called mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2). Together, these complexes coordinate a variety of processes that include protein translation, autophagy, proliferation, survival and metabolism in response to nutrient, energy and growth factor signals. Consistent with its role as a growth-promoting pathway, numerous studies have found that Mtor signaling is hyper-activated in a broad spectrum of human cancers. In particular, mTORC2 is considered a primary effector of the phosphatidylinositol-3-kinase (PI3K) signaling pathway, which is mutated in a majority of human cancers, in part through its ability to phosphorylate and regulate the proto-oncogene Akt/PKB. Many biological functions of mTOR have been pharmacologically explored using the natural product rapamycin, an allosteric inhibitor that has been reviewed extensively elsewhere. This review will focus specifically on the development of small molecule ATP-competitive inhibitors of mTOR and their prospects as a targeted therapy.
mTOR; PI3K; Cancer
Cancer is a multifaceted disease that results from dysregulated normal cellular signaling networks caused by genetic, genomic and epigenetic alterations at cell or tissue levels. Uncovering the underlying protein signaling network changes, including cell cycle gene networks in cancer, aids in understanding the molecular mechanism of carcinogenesis and identifies the characteristic signaling network signatures unique for different cancers and specific cancer subtypes. The identified signatures can be used for cancer diagnosis, prognosis, and personalized treatment. During the past several decades, the available technology to study signaling networks has significantly evolved to include such platforms as genomic microarray (expression array, SNP array, CGH array, etc.) and proteomic analysis, which globally assesses genetic, epigenetic, and proteomic alterations in cancer. In this review, we compared Pathway Array analysis with other proteomic approaches in analyzing protein network involved in cancer and its utility serving as cancer biomarkers in diagnosis, prognosis and therapeutic target identification. With the advent of bioinformatics, constructing high complexity signaling networks is possible. As the use of signaling network-based cancer diagnosis, prognosis and treatment is anticipated in the near future, medical and scientific communities should be prepared to apply these techniques to further enhance personalized medicine.
Lineage-specific DNA-binding transcription factors regulate development by activating and repressing particular set of genes required for the acquisition of a specific cell type. Pax6 is a paired domain and homeodomain-containing transcription factor essential for development of central nervous, olfactory and visual systems, as well as endocrine pancreas. Haploinsufficiency of Pax6 results in perturbed lens development and homeostasis. Loss-of-function of Pax6 is incompatible with lens lineage formation and results in abnormal telencephalic development. Using DNA microarrays, we have identified 559 genes expressed differentially between 1-day old mouse Pax6 heterozygous and wild type lenses. Of these, 178 (31.8%) were similarly increased and decreased in Pax6 homozygous embryonic telencephalon [Holm PC, Mader MT, Haubst N, Wizenmann A, Sigvardsson M, Götz M (2007) Loss- and gain-of-function analyses reveals targets of Pax6 in the developing mouse telencephalon. Mol Cell Neurosci 34: 99–119]. In contrast, 381 (68.2%) genes were differently regulated between the lens and embryonic telencephalon. Differential expression of nine genes implicated in lens development and homeostasis: Cspg2, Igfbp5, Mab21l2, Nrf2f, Olfm3, Spag5, Spock1, Spon1 and Tgfb2, was confirmed by quantitative RT-PCR, with five of these genes: Cspg2, Mab21l2, Olfm3, Spag5 and Tgfb2, identified as candidate direct Pax6 target genes by quantitative chromatin immunoprecipitation (qChIP). In Mab21l2 and Tgfb2 promoter regions, twelve putative individual Pax6-binding sites were tested by electrophoretic mobility shift assays (EMSAs) with recombinant Pax6 proteins. This led to the identification of two and three sites in the respective Mab21l2 and Tgfb2 promoter regions identified by qChIPs. Collectively, the present studies represent an integrative genome-wide approach to identify downstream networks controlled by Pax6 that control mouse lens and forebrain development.
The prediction of RNA secondary structure can be facilitated by incorporating with comparative analysis of homologous sequences. However, most of existing
comparative methods are vulnerable to alignment errors and thus are of low accuracy in practical application. Here we improve the prediction of RNA
secondary structure by detecting and assessing conserved stems shared by all sequences in the alignment. Our method can be summarized by: 1) we detect possible
stems in single RNA sequence using the so-called position matrix with which some possibly paired positions can be uncovered; 2) we detect conserved stems across
multiple RNA sequences by multiplying the position matrices; 3) we assess the conserved stems using the Signal-to-Noise; 4) we compute the optimized secondary
structure by incorporating the so-called reliable conserved stems with predictions by RNAalifold program. We tested our method on data sets of RNA alignments with
known secondary structures. The accuracy, measured as sensitivity and specificity, of our method is greater than predictions by RNAalifold.
RNA; secondary structure; conserved stem; homologous sequence; Signal-to-Noise
Besides linear RNAs, pre-mRNA splicing generates three forms of RNAs: lariat introns, Y-structure introns from trans-splicing, and circular exons through exon skipping. To study the persistence of excised introns in total cellular RNA, we used three Escherichia coli 3′ to 5′ exoribonucleases. Ribonuclease R (RNase R) thoroughly degrades the abundant linear RNAs and the Y-structure RNA, while preserving the loop portion of a lariat RNA. Ribonuclease II (RNase II) and polynucleotide phosphorylase (PNPase) also preserve the lariat loop, but are less efficient in degrading linear RNAs. RNase R digestion of the total RNA from human skeletal muscle generates an RNA pool consisting of lariat and circular RNAs. RT–PCR across the branch sites confirmed lariat RNAs and circular RNAs in the pool generated by constitutive and alternative splicing of the dystrophin pre-mRNA. Our results indicate that RNase R treatment can be used to construct an intronic cDNA library, in which majority of the intron lariats are represented. The highly specific activity of RNase R implies its ability to screen for rare intragenic trans-splicing in any target gene with a large background of cis-splicing. Further analysis of the intronic RNA pool from a specific tissue or cell will provide insights into the global profile of alternative splicing.
Exonic splicing enhancers (ESEs) are pre-mRNA cis-acting elements required for splice-site recognition. We previously developed a web-based program called ESEfinder that scores any sequence for the presence of ESE motifs recognized by the human SR proteins SF2/ASF, SRp40, SRp55 and SC35 (). Using ESEfinder, we have undertaken a large-scale analysis of ESE motif distribution in human protein-coding genes. Significantly higher frequencies of ESE motifs were observed in constitutive internal protein-coding exons, compared with both their flanking intronic regions and with pseudo exons. Statistical analysis of ESE motif frequency distributions revealed a complex relationship between splice-site strength and increased or decreased frequencies of particular SR protein motifs. Comparison of constitutively and alternatively spliced exons demonstrated slightly weaker splice-site scores, as well as significantly fewer ESE motifs, in the alternatively spliced group. Our results underline the importance of ESE-mediated SR protein function in the process of exon definition, in the context of both constitutive splicing and regulated alternative splicing.
An investigation of how to improve mammalian promoter prediction by incorporating both transcript and conservation information leads to the creation of CSHLmpd, a mammalian promoter database.
Large-scale and high-throughput genomics research needs reliable and comprehensive genome-wide promoter annotation resources. We have conducted a systematic investigation on how to improve mammalian promoter prediction by incorporating both transcript and conservation information. This enabled us to build a better multispecies promoter annotation pipeline and hence to create CSHLmpd (Cold Spring Harbor Laboratory Mammalian Promoter Database) for the biomedical research community, which can act as a starting reference system for more refined functional annotations.