Analysis of intronless genes involved in oscillation and differentiation

The genomes of higher eukaryotes are replete with intron-containing genes. Transcription of these genes produces precursor mRNAs containing intervening sequences, which are subsequently removed and the exons spliced together to form the mature mRNA. However, a small proportion of eukaryotic protein-coding genes are intronless and therefore bypass post-transcriptional splicing events. Although a large proportion of intronless genes are known to code for certain types of proteins, their specific role in the genome of higher organism is perplexing. This research set out to elucidate the functions of intronless genes in humans by studying their involvement in the expression pattern of oscillatory gene that occurs in the pre-somitic mesoderm of developing embryo. Twenty-seven (27) human homologs of mouse oscillatory genes were analysed to determine the number of exons present in them using various bioinformatics databases. The result obtained identified two intronless genes –NRARP and ID1 – which are associated with the Notch signalling pathway of the segmentation clock. This represented 7.4% of the total oscillatory genes analysed. No intronless gene was found in the Wnt and FGF signalling pathways – two other pathways famous for oscillatory gene expression. The proteins encoded by the intronless genes are involved in several important biological processes including angiogenesis, cell cycle control and in the regulation of cellular senescence. Although oscillatory genes had fewer numbers of introns compared to the non-oscillatory genes, the intronless genes were not implicated in the regulation of the precise timing events of the segmentation clock. This result may also point to the fact that the rapid expression rate of the oscillatory genes in the PSM may favour the reduced intron length of the oscillatory genes.

Phosphor-IWS1-dependent U2AF2 splicing regulates trafficking of CAR-E-positive intronless gene mRNAs and sensitivity to viral infection

AKT-phosphorylated IWS1 promotes Histone H3K36 trimethylation and alternative RNA splicing of target genes, including the U2AF65 splicing factor-encoding U2AF2. The predominant U2AF2 transcript, upon IWS1 phosphorylation block, lacks the RS-domain-encoding exon 2, and encodes a protein which fails to bind Prp19. Here we show that although both U2AF65 isoforms bind intronless mRNAs containing cytoplasmic accumulation region elements (CAR-E), only the RS domain-containing U2AF65 recruits Prp19 and promotes their nuclear export. The loading of U2AF65 to CAR-Elements was RS domain-independent, but RNA PolII-dependent. Virus- or poly(I:C)-induced type I IFNs are encoded by genes targeted by the pathway. IWS1 phosphorylation-deficient cells therefore, express reduced levels of IFNα1/IFNβ1 proteins, and exhibit enhanced sensitivity to infection by multiple cytolytic viruses. Enhanced sensitivity of IWS1-deficient cells to Vesicular Stomatitis Virus and Reovirus resulted in enhanced apoptotic cell death via caspase activation. Inhibition of this pathway may therefore sensitize cancer cells to oncolytic viruses.

FOXE1 polymorphism rs965513 predisposes to thyroid cancer in a European cohort

Objective: FOXE1 is an intronless gene on chromosome 9 which plays a significant role in thyroid morphogenesis. Mutations in FOXE1 are associated with thyroid phenotypes including congenital hypothyroidism, thyroid dysgenesis and thyroid cancer. This study aims to investigate the frequency and impact of a single nucleotide polymorphism(rs965513, G>A) at 9q22.23 in a Western European cohort of patients with differentiated thyroid cancer(DTC), compared to controls. Design: This is a candidate gene case control study. Methods: 277 patients with histologically confirmed DTC were recruited from tertiary referral centres in Ireland and France. 309 cancer-free controls were recruited from the community. DNA was extracted from buccal swabs or whole blood of control subjects and patients with DTC. Allelic and genotypic frequencies among patients were compared with controls, to assess the risk for disease conferred by homozygous and heterozygous carriers compared to wild-type genotypes. Genotyping was performed using Taqman-based PCR. Results: 277 patients with confirmed DTC and 309 non-cancer controls were genotyped. The frequency of the minor allele among cases was 0.45 compared to 0.34 among controls. The genotypic odds ratio for heterozygotes was 1.66(CI 1.16-2.39, p=0.00555), increasing to 2.93(CI 1.70-5.05, p=0.00007) for rare homozygotes. All subjects were in Hardy-Weinberg equilibrium(X2, p=0.09, p=0.07 respectively). Conclusions: This FOXE1 polymorphism is a low penetrance variant associated with DTC susceptibility in this cohort. The minor allele was identified among patients with thyroid cancer significantly more frequently than controls. An allele dosage effect was observed, with rare homozygous genotypes conferring greater risk than heterozygotes.

Biochemical, structural insights of newly isolated AA16 family of Lytic Polysaccharide Monooxygenase (LPMO) from Aspergillus fumigatus and investigation of its synergistic effect using biomass.

The efficient conversion of lignocellulosic biomass into fermentable sugar is a bottleneck for the cheap production of bio-ethanol. The recently identified enzyme Lytic Polysaccharide Monooxygenase (LPMO) family has brought new hope because of its boosting capabilities of cellulose hydrolysis. In this report, we have identified and characterized a new class of auxiliary (AA16) oxidative enzyme LPMO from the genome of a locally isolated thermophilic fungus Aspergillus fumigatus (NITDGPKA3) and evaluated its boosting capacity of biomass hydrolysis. The AfLPMO16 is an intronless gene and encodes the 29kDa protein. While Sequence-wise, it is close to the C1 type of AaAA16 and cellulose-active AA10 family of LPMOs, but the predicted three-dimensional structure shows the resemblance with the AA11 family of LPMO (PDB Id: 4MAH). The gene was expressed under an inducible promoter (AOX1) with C-terminal His tag in the Pichia pastoris. The protein was purified using Ni-NTA affinity chromatography, and we studied the enzyme kinetics with 2,6-dimethoxyphenol. We observed polysaccharides depolymerization activity with Carboxymethyl cellulose (CMC) and Phosphoric acid swollen cellulose (PASC). Moreover, the simultaneous use of cellulase cocktail (commercial) and AfLPMO16 enhances lignocellulosic biomass hydrolysis by 2-fold, which is highest so far reported in the LPMO family.

Improved cold tolerance in switchgrass by a novel CCCH-type zinc finger transcription factor gene, PvC3H72, associated with ICE1–CBF–COR regulon and ABA-responsive genes

Background Switchgrass (Panicum virgatum) is a warm-season perennial grass. Improving its cold tolerance is important for its sustainable production in cooler regions. Through genome-wide bioinformatic analysis of switchgrass Zinc finger-CCCH genes (PvC3Hs), we found that several PvC3Hs, including PvC3H72, might play regulatory roles in plant cold tolerance. The objectives of this study were to characterize PvC3H72 using reverse genetics approach and to understand its functional role in cold signal transduction and cold tolerance in switchgrass. Results PvC3H72 is an intronless gene encoding a transcriptional activation factor. The expression of PvC3H72 was rapidly and highly induced by cold stress. Transgenic switchgrass with over-expressed PvC3H72 driven under maize ubiquitin promoter showed significantly improved chilling tolerance at 4 °C as demonstrated by less electrolyte leakage and higher relative water content than wild-type (WT) plants, as well as significantly higher survival rate after freezing treatment at − 5 °C. Improved cold tolerance of PvC3H72 transgenic lines was associated with significantly up-regulated expression of ICE1–CBF–COR regulon and ABA-responsive genes during cold treatment. Conclusions PvC3H72 was the first characterized switchgrass cold-tolerance gene and also the only Znf-CCCH family gene known as a transcription factor in plant cold tolerance. PvC3H72 was an added signaling component in plant cold tolerance associated with regulation of ICE1–CBF–COR regulon and ABA-responsive genes. Knowledge gained in this study not only added another acting component into plant cold-tolerance mechanism, but also be of high value for genetic improvement of cold tolerance in switchgrass as well as other warm-season grasses.

Ovarian cancer G protein-coupled receptor 1 inhibits A549 cells Migration through Casein kinase 2α intronless gene

We have previously reported that ovarian cancer G protein-coupled receptor 1 (OGR1) is a new metastasis suppressor gene. We have also reported for the first time that a new intronless gene for casein kinase 2α (CSNK2A3) is expressed in human cells. The promoter of the well-known casein kinase 2α (CSNK2A1) displays characteristics of housekeeping gene whereas CSNK2A3 has a characteristic of a regulated promoter with two TATA boxes and a CAAT box. In this study, we found that OGR1 up-regulates expression of CSNK2A3 by about 3 folds in A549 cells but not CSNK2A1. OGR1 also up-regulates expression of neutral endopeptidase (NEP). The OGR1 induced inhibition of A549 cell migration is completely abrogated by inhibition of casein kinase 2α activity, whereas partial abrogation (~ 30%) was observed in the presence of NEP inhibition. The results also revealed that OGR1 regulates CSNK2A3 via activation of Rac1/cdc42 and MAPKs pathways. CK2 is ubiquitously expressed and in contrast it is believed to be a constitutively active enzyme and its regulation appears to be independent of known second messengers. There is no previous report on how expression of CK2α in cancer cells is regulated although many studies have report of aberrant expression of the kinase in cancer. In the current study, we are reporting for the first time the regulation of intronless casein kinase 2α gene, CSNK2A3 in cancer cells. Our findings suggest that the aberrantly casein kinase 2α expression found in various cancer cells may the due to CSNK2A3 expression which is potentially regulated by several master regulators of the developmental pathways rather than well-known casein kinase 2α gene, CSNK2A1.

Characterization of strawberry (Fragaria vesca) sequence genome

BackgroundIn order to understand strawberry genes’ structure and evolution in this era of genomics, it is important to know the general statistical characteristics of the gene, intron and exon structures of strawberry and the expression of genes on different parts of strawberry genome. In the present study, about 32,422 genes on strawberry chromosomes were evaluated, and a number of bioinformatic softwares were used to analyze the characteristics of genes, exons and introns, expression of genes in different regions on the chromosomes. Also, the positions of strawberry centromeres were predicted.ResultsOur results showed that, there are differences in the various features of different chromosomes and also vary in different parts of the same chromosome. The longer the number of genes, the longer the length of chromosome. The average length of genes is about 2809bp and the length of the individual gene is 0–2000bp with 5.3 exons and 4.3 introns per gene. The average length of the exon was 229bp and the intron was 413bp. Among the evaluated genes, ehe intronless gene accounted for 20.05%. Consistently a same trend with the expression levels of the same parts of the gene on a chromosome in different organizations was observed. Finally, the number of genes was positively correlated with the number of intronless, and there was a negative correlation of the length of the gene. The length of the gene depends primarily on the length of the intron, and the length of the exon has little effect on it. The number of exons was negatively correlated with the length of the exons, and the intron was also true.ConclusionThe results of this investigation could definitely provide a significant foundation for further research on function analysis of gene family in Strawberry.

Enhanced at Puberty-1 (Eap1) Expression Critically Regulates the Onset of Puberty Independent of Hypothalamic Kiss1 Expression

Background/Aims: Enhance at puberty-1 (Eap1) is an intronless gene that regulates the onset of puberty through a network of hypothalamic genes. However, precise mechanistic events essential for Eap1-regulation of puberty have not been fully elucidated. Eap1 is thought to promote the initiation of puberty through regulation of the hypothalamic metastasis-suppressor KiSS1. We aim to investigate this hypothesis by genetically perturbing Eap1 through RNA interference in vivo. Methods: We first engineered and optimized four sets of shRNAs that target rat Eap1 mRNA as well as one negative control shRNA. After generating lentiviral (LV) particles, we examined the suppression of Eap1 in NRK-54E cell line to select the most efficient one. Sequencelly, LV-Eap1-shRNA or controls including LV-eGFP and saline were intraventricular microinjected into 21-day-old rats. Rats were raised in appropriate conditions and we examined the time of vaginal opening, ovary physiology as well as hypothalamic puberty-regulatory genes at three developmental stages: juvenile (postnatal day PND25), early puberty (PND35), adult (PND42). Results: Hypothalamic suppression of Eap1 delayed the onset of rat vaginal opening. Hematoxylin and eosin (H&E) staining revealed a significant reduction of corpus luteum (CL) at PND35, but at PND42 CL levels were normal relative to control. In conjunction with differences in phenotype and ovary morphology, GnRH expression and transcripts were also reduced at PND25 and PND35, while their levels were similar to control at PND42. KiSS1 mRNA and protein levels were not significantly different at all three developmental stages. Conclusion: Eap1 expression critically regulates puberty as well as GnRH expression. However, Eap1-regulation of puberty may not necessitate KiSS1/GPR54 signaling.

Novel pH-Stable Glycoside Hydrolase Family 3 β-Xylosidase from Talaromyces amestolkiae: an Enzyme Displaying Regioselective Transxylosylation

ABSTRACTThis paper reports on a novel β-xylosidase from the hemicellulolytic fungusTalaromyces amestolkiae. The expression of this enzyme, called BxTW1, could be induced by beechwood xylan and was purified as a glycoprotein from culture supernatants. We characterized the gene encoding this enzyme as an intronless gene belonging to the glycoside hydrolase gene family 3 (GH3). BxTW1 exhibited transxylosylation activity in a regioselective way. This feature would allow the synthesis of oligosaccharides or other compounds not available from natural sources, such as alkyl glycosides displaying antimicrobial or surfactant properties. Regioselective transxylosylation, an uncommon combination, makes the synthesis reproducible, which is desirable for its potential industrial application. BxTW1 showed high pH stability and Cu2+tolerance. The enzyme displayed a pI of 7.6, a molecular mass around 200 kDa in its active dimeric form, andKmandVmaxvalues of 0.17 mM and 52.0 U/mg, respectively, using commercialp-nitrophenyl-β-d-xylopyranoside as the substrate. The catalytic efficiencies for the hydrolysis of xylooligosaccharides were remarkably high, making it suitable for different applications in food and bioenergy industries.