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Results for “exon dynamics”

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PubMed2026

Identification and validation of an alternatively spliced novel isoform of maspin that modulate genes involved in inflammatory and apoptotic pathways.

Maspin regulates cellular adhesion, migration, apoptosis, angiogenesis, and tumor suppression in a tissue and context-dependent manner. Its functional diversity is governed largely thorough extracellular matrix interactions, subcellular localization, and the reactive center loop (RCL), although the structural details are not well understood. To examine whether alternative splicing contributes to this heterogeneity, we analysed the SERPINB5 gene using a computational genomics approach and identified a novel 80 bp coding exon upstream of the first coding exon (E1). The alternatively spliced transcript was validated in human skin and esophagus by semi-nested touchdown PCR, quantitative real-time PCR, and Sanger sequencing. Recombinant B5N displayed a red-shifted fluorescence emission spectrum, indicating a more solvent-exposed conformation, which was supported by molecular dynamics simulations showing greater exposure of the nuclear localization signal (NLS) and the reactive center loop. Enzyme kinetic assays demonstrated concentration-dependent enhancement of tissue plasminogen activator (tPA) activity by both isoforms. In HaCaT cells, wildtype maspin produced stronger antiproliferative and anti-migratory effects, whereas B5N was only mildly antiproliferative. Annexin V/7-AAD staining revealed that wildtype maspin induced higher early apoptosis and cell death, while B5N produced lower overall cell death but a greater proportion of late apoptotic cells. RNA-seq of transfected HaCaT cells identified differentially expressed genes enriched in inflammatory, antiviral, and apoptotic pathways, which was validated by qPCR, and several of these were markedly upregulated in SARS-CoV-2 infected A549 cells. Thus, a novel N-terminally extended maspin isoform with differentially regulated gene profile is identified and validated in this study.

Apoptosis↗

PubMed2004

Potentiation of myelin proteolipid protein (Plp) gene expression is mediated through AP-1-like binding sites.

The myelin proteolipid protein (Plp) gene is expressed in oligodendrocytes and encodes the most abundant protein found in mature CNS myelin. Expression of the gene is dynamic and peaks during the active myelination period of CNS development. The surge in Plp gene activity during this period has been purported to be mediated by a positive regulatory region located within the first intron. This region, designated ASE for antisilencer/enhancer, is located approximately 1 kb downstream of exon 1 sequences and encompasses nearly 100 bp. However, neither the critical nucleotides within this region, nor the associated DNA-binding proteins have been identified. In the present study, DNase I footprinting analysis demonstrated widespread protection of the region on both the coding and non-coding strands suggesting that multiple transcription factors are likely involved. Targeting of putative DNA-protein binding sites contained within the ASE by gel shift, transfection and mutagenesis studies revealed the importance of several AP-1-like binding sites in governing high levels of Plp gene expression in oligodendrocytes. Our results suggest that factors, which bind to these sites, form the core of a multiprotein complex that assembles on the ASE and ultimately affects the temporal regulation of the gene in oligodendrocytes.

Animals↗

PubMed1990

Developmental changes in levels of proopiomelanocortin intron A-containing heterogeneous nuclear RNA and mature messenger RNA in the anterior and neurointermediate lobes of the rat pituitary.

The POMC cells of the rat pituitary undergo dynamic phenotypic changes during differentiation. Here we have determined that alterations in the relative levels of a POMC precursor RNA species and POMC mRNA occur during development and may represent another level at which the POMC phenotype is developmentally regulated. We performed solution hybridization/nuclease protection assays using a POMC exon 1/intron A splice junction probe to quantitate levels of both intron A-containing POMC heterogeneous nuclear (hnRNA) and fully processed POMC mRNA in separated anterior and neurointermediate lobes of the fetal, neonatal, and adult pituitary. The levels of POMC hnRNA per anterior lobe increased 7-fold from embryonic day 15 to adulthood (0.022 to 0.159 fmol/lobe), while POMC mRNA levels increased 121-fold (0.15 to 18.2 fmol/lobe). POMC hnRNA levels per neurointermediate lobe increased 23-fold from embryonic day 18 to adulthood (0.024 to 0.54 fmol/lobe), while POMC mRNA levels increased 69-fold (0.65 to 44.6 fmol/lobe). Thus, both anterior and neurointermediate lobes contain higher relative abundances of POMC hnRNA compared to mRNA during early development. These subsequently decrease (from 1:7 to 1: approximately 110 in the anterior lobe and from 1:27 to 1:83 in the intermediate lobe over the ages examined) as the levels of POMC mRNA in both anterior and neurointermediate lobe increase at a greater rate than POMC hnRNA as development progresses. These results provide the first measurements of POMC mRNA and hnRNA levels during ontogeny and suggest that there may be a developmental change in the regulation of POMC primary transcript processing.

Animals↗

PubMed1996

Splicing of precursors to mRNA in higher plants: mechanism, regulation and sub-nuclear organisation of the spliceosomal machinery.

The removal of introns from pre-mRNA transcripts and the concomitant ligation of exons is known as pre-mRNA splicing. It is a fundamental aspect of constitutive eukaryotic gene expression and an important level at which gene expression is regulated. The process is governed by multiple cis-acting elements of limited sequence content and particular spatial constraints, and is executed by a dynamic ribonucleoprotein complex termed the spliceosome. The mechanism and regulation of pre-mRNA splicing, and the sub-nuclear organisation of the spliceosomal machinery in higher plants is reviewed here. Heterologous introns are often not processed in higher plants indicating that, although highly conserved, the process of pre-mRNA splicing in plants exhibits significant differences that distinguish it from splicing in yeast and mammals. A fundamental distinguishing feature is the presence of and requirement for AU or U-rich intron sequence in higher-plant pre-mRNA splicing. In this review we document the properties of higher-plant introns and trans-acting spliceosomal components and discuss the means by which these elements combine to determine the accuracy and efficiency of pre-mRNA processing. We also detail examples of how introns can effect regulated gene expression by affecting the nature and abundance of mRNA in plants and list the effects of environmental stresses on splicing. Spliceosomal components exhibit a distinct pattern of organisation in higher-plant nuclei. Effective probes that reveal this pattern have only recently become available, but the domains in which spliceosomal components concentrate were identified in plant nuclei as enigmatic structures some sixty years ago. The organisation of spliceosomal components in plant nuclei is reviewed and these recent observations are unified with previous cytochemical and ultrastructural studies of plant ribonuleoprotein domains.

Cell Nucleus↗

PubMed2000

Dynamic regulation of neuronal NO synthase transcription by calcium influx through a CREB family transcription factor-dependent mechanism.

Neuronal nitric oxide (NO) synthase (nNOS) is dynamically regulated in response to a variety of physiologic and pathologic stimuli. Although the dynamic regulation of nNOS is well established, the molecular mechanisms by which such diverse stimuli regulate nNOS expression have not yet been identified. We describe experiments demonstrating that Ca(2+) entry through voltage-sensitive Ca(2+) channels regulates nNOS expression through alternate promoter usage in cortical neurons and that nNOS exon 2 contains the regulatory sequences that respond to Ca(2+). Deletion and mutational analysis of the nNOS exon 2 promoter reveals two critical cAMP/Ca(2+) response elements (CREs) that are immediately upstream of the transcription start site. CREB binds to the CREs within the nNOS gene. Mutation of the nNOS CREs as well as blockade of CREB function results in a dramatic loss of nNOS transcription. These findings suggest that nNOS is a Ca(2+)-regulated gene through the interactions of CREB on the CREs within the nNOS exon 2 promoter and that these interactions are likely to be centrally involved in the regulation of nNOS in response to neuronal injury and activity-dependent plasticity.

Animals↗

PubMed2006

Comparative analysis of the paired immunoglobulin-like receptor (PILR) locus in six mammalian genomes: duplication, conversion, and the birth of new genes.

Manyaspects of the immune system are controlled by homologous cell surface receptors that mediate inhibitory and activating pathways. The paired immunoglobulin-like receptor (PILR) locus at 7q22 encodes both PILRA, an inhibitory receptor, and PILRB, its activating counterpart. Mouse Pilrb1 is a novel immune system regulator, and its ligand Cd99 participates in the recruitment of T-cells to inflamed tissue. We characterized the PILR locus in six mammalian genomes and investigated the structure and mRNA expression of human PILRB. Synteny at the PILR locus is conserved in the human, chimpanzee, dog, mouse and rat genomes. The absence of the PILR locus in opossum and chicken genomes suggests it arose after the divergence of placental and nonplacental mammals. In humans, a Williams-Beuren syndrome-related segmental duplication has created a complex chimeric transcript representing the predominantly expressed form of PILRB. Unlike PILRA, PILRB transcripts were detected in a wide variety of tissues including cells of the lymphoid lineage. In the mouse genome, a second activating gene, Pilrb2, and six pseudogenes were found. Extensive gene duplications in the rat genome have resulted in at least 27 Pilrb genes and or pseudogenes. Abundant gene duplication events involving novel CD99-related genes were also detected in the rat genome. In addition to duplication, we show that gene conversion has played a persistent role in the evolution of the PILR genes. Overall, we demonstrate that the PILR locus is dynamically evolving via multiple evolutionary mechanisms in several mammalian genomes.

Animals↗

PubMed2002

Genomic organization of profilin-III and evidence for a transcript expressed exclusively in testis.

Profilins are small, widely expressed actin binding proteins, thought to be key regulators of actin dynamics in living cells. So far, three profilin-genes have been described: profilin-I (PFN1), profilin-II (PFN2) with two splice variants and the recently identified profilin-III (PFN3). Here we describe the genomic organization of the genes encoding human and mouse profilin-III. Both are single exon genes and lie in close vicinity to the renal sodium-phosphate transport gene 2 (SLC34A1, NPT2) which is highly expressed in kidney. Northern hybridization to rat tissues has previously demonstrated expression of an approximately 4.5 kb long profilin-III mRNA transcript in kidney and a mRNA transcript of approximately 1 kb in length in testis. Here we show that mouse profilin-III expression is restricted to testis and that the 4.2 kb profilin-III mRNA in kidney is the result of a slc34a1 transcript which includes the antisense profilin-III open reading frame in its 3'-untranslated region. Finally, we demonstrate by in situ hybridization that profilin-III mRNA is localized to cells in the late stage of spermatogenesis.

Amino Acid Sequence↗

PubMed2003

Cancer and epigenesis: a developmental perspective.

Traditionally, molecular geneticists have studied the genome, that is, DNA. This has included its sequencing, with identification of promoters, enhancers, introns, exons, and mutations. During the last 10 to 15 years, it has become clear that this study of "naked" DNA imposed major limitations on our understanding of gene regulation, and that DNA must be studied in conjunction with its protein backbone (chromatin). This new vision has shown that chromatin is a very dynamic molecule, and that changes in DNA methylation, in histones leg, their localization, density, and whether they are acetylated, methylated, phosphorylated and/or ubiquitinated), and in the Polycomb-Trithorax equilibrium are all crucial for the control of gene expression. Furthermore, it was found that like DNA, chromatin-related proteins and DNA methylation are generally passed on unchanged from one cell to its daughters. Thus, gene expression is partly controlled through chromatin modifications that are transmitted from one cell to all its descendants. Such a control is referred to as "epigenetic," as the DNA sequence is not altered. Hence, these epigenetic "marks" can be erased in the early embryo, rendering these cells totipotent. This chapter presents a basic overview of epigenesis in the control of normal embryonal cell differentiation, including imprinting. Similarities between embryonal and cancer cells are highlighted, and the potential impact of chemotherapy aimed at epigenetic mechanisms is reviewed.

Alleles↗

PubMed2005

Stage-by-stage change in DNA methylation status of Dnmt1 locus during mouse early development.

Methylation of DNA is involved in tissue-specific gene control, and establishment of DNA methylation pattern in the genome is thought to be essential for embryonic development. Three isoforms of Dnmt1 (DNA methyltransferase 1) transcripts, Dnmt1s, Dnmt1o, and Dnmt1p, are produced by alternative usage of multiple first exons. Dnmt1s is expressed in somatic cells. Dnmt1p is found only in pachytene spermatocytes, whereas Dnmt1o is specific to oocytes and preimplantation embryos. Here we determined that there is a tissue-dependent differentially methylated region (T-DMR) in the 5' region of Dnmt1o but not in that of the Dnmt1s/1p. The methylation status of the Dnmt1o T-DMR was distinctively different in the oocyte from that in the sperm and adult somatic tissues and changed at each stage from fertilization to blastocyst stage, suggesting that active methylation and demethylation occur during preimplantation development. The T-DMR was highly methylated in somatic cells and embryonic stem cells. Analysis using Dnmt-deficient embryonic stem cell lines revealed that Dnmt1, Dnmt3a, and Dnmt3b are each partially responsible for maintenance of methylation of Dnmt1o T-DMR. In particular, there are compensatory and cooperative roles between Dnmt3a and Dnmt3b. Thus, the regulatory region of Dnmt1o, but not of Dnmt1s/1p, appeared to be a target of DNA methylation. The present study also suggested that the DNA methylation status of the gene region dynamically changes during embryogenesis independently of the change in the bulk DNA methylation status.

5' Untranslated Regions↗

PubMed2001

Dynamic DNA methylation change in the CpG island region of p15 during human myeloid development.

We examined the kenetics of p15 methylation and expression during myeloid development. We treated human cord blood CD34+ cells with either GM-CSF alone or in combination with stem cell factor and followed methylation at this locus using bisulfite genomic sequencing. CD34+ cells were found to be either fully methylated or completely unmethylated at 27 CpG dinucleotide sites in exon 1 and at 18 CpG sites in the promoter region of the p15 gene. A time-course study showed that the percentage of the allelic methylation of p15 CpG island increased to approximately 50% to 60% until 7 days after cytokine stimulation, then decreased to less than 10% after 21 days. The methylation was also observed in bone marrow CD34+ cells exposed to GM-CSF. p15 expression varied inversely with methylation. Expression was negligible or at low levels until 14 days, after which it increased substantially. The frequency of myeloid colony-forming cells in the progeny decreased and myeloid-specific markers increased in the later stages. Based on our observations on cells grown with GM-CSF and 5-aza-2'-deoxycytidine, DNA methylation of the p15 promoter region CpG island appears to be associated with proliferation rather than differentiation of normal human myeloid progenitors.

Alleles↗

PubMed2006

On the abundance, amino acid composition, and evolutionary dynamics of low-complexity regions in proteins.

Protein sequences frequently contain regions composed of a reduced number of amino acids. Despite their presence in about half of all proteins and their unusual prevalence in the malaria parasite Plasmodium falciparum, the function and evolution of such low-complexity regions (LCRs) remain unclear. Here we show that LCR abundance and amino acid composition depend largely, but not exclusively, on genomic A+T content and obey power-law growth dynamics. Further, our results indicate that LCRs are analogous to microsatellites in that DNA replication slippage and unequal crossover recombination are important molecular mechanisms for LCR expansion. We support this hypothesis by demonstrating that the size of LCR insertions/deletions among orthologous genes depends upon length. Moreover, we show that LCRs enable intra-exonic recombination in a key family of cell-surface antigens in P. falciparum and thus likely facilitate the generation of antigenic diversity. We conclude with a mechanistic model for LCR evolution that links the pattern of LCRs within P. falciparum to its high genomic A+T content and recombination rate.

Amino Acids↗

PubMed1995

A long purine-pyrimidine homopolymer acts as a transcriptional diode.

Polypurine-polypyrimidine (R.Y) sequences have the unusual ability to form DNA triple helices. Such tracts are overrepresented upstream of eukaryotic genes, although a function there has not been clear. We report that transcription in vitro into one such upstream R.Y tract in the direction that makes a predominantly purine RNA is effectively blocked by formation of an intramolecular triple helix. The triplex is triggered by transcription and stabilized by the binding of nascent purine RNA to the template. Transcription in the opposite direction is not restricted. Polypurine-polypyrimidine DNA may provide a dynamic and selective block to transcription without the aid of accessory proteins.

Amino Acid Sequence↗

PubMed2003

Haplotype block partition with limited resources and applications to human chromosome 21 haplotype data.

Recent studies have shown that the human genome has a haplotype block structure such that it can be decomposed into large blocks with high linkage disequilibrium (LD) and relatively limited haplotype diversity, separated by short regions of low LD. One of the practical implications of this observation is that only a small fraction of all the single-nucleotide polymorphisms (SNPs) (referred as "tag SNPs") can be chosen for mapping genes responsible for human complex diseases, which can significantly reduce genotyping effort, without much loss of power. Algorithms have been developed to partition haplotypes into blocks with the minimum number of tag SNPs for an entire chromosome. In practice, investigators may have limited resources, and only a certain number of SNPs can be genotyped. In the present article, we first formulate this problem as finding a block partition with a fixed number of tag SNPs that can cover the maximal percentage of the whole genome, and we then develop two dynamic programming algorithms to solve this problem. The algorithms are sufficiently flexible to permit knowledge of functional polymorphisms to be considered. We apply the algorithms to a data set of SNPs on human chromosome 21, combining the information of coding and noncoding regions. We study the density of SNPs in intergenic regions, introns, and exons, and we find that the SNP density in intergenic regions is similar to that in introns and is higher than that in exons, results that are consistent with previous studies. We also calculate the distribution of block break points in intergenic regions, genes, exons, and coding regions and do not find any significant differences.

Chromosomes, Human, Pair 21↗

PubMed2005

Transcription of the murine gammaherpesvirus 68 ORF73 from promoters in the viral terminal repeats.

Gammaherpesviruses persist as latent episomes in a dynamic lymphocyte pool. The regulated production of an episome maintenance protein is therefore crucial to their survival. The transcription initiation site of the murine gammaherpesvirus 68 episome maintenance protein, ORF73, was mapped to the viral terminal repeats, more than 10 kb distant from the open reading frame (ORF) itself. A 5' non-coding exon in the terminal repeats was spliced to the right end of the viral unique sequence, and then across ORFs 75a, 75b, 75c and 74 to ORF73. The right-hand portion of a single repeat unit was sufficient for constitutive promoter activity. The unique left end of the viral genome further enhanced ORF73 transcription. This, together with the large size of the predominant ORF73 mRNA, suggested that transcription initiates in distal repeat units and then splices between repeats to generate an extensive 5' untranslated region. A second promoter in the left-hand portion of the proximal terminal repeat unit generated a transcript which overlapped that of ORF73, but failed to splice to the ORF73 coding exon and so transcribed ORF75a. In distal repeat copies, however, transcription from this promoter would enter the next repeat unit to become an ORF73 mRNA. There was a third promoter just upstream of ORF73 itself. These data indicate that ORF73 transcription is highly complex, and support the idea that the terminal repeats of gamma-2-herpesviruses constitute a vital component of episomal persistence.

Animals↗

PubMed2006

Improving gene annotation using peptide mass spectrometry.

Annotation of protein-coding genes is a key goal of genome sequencing projects. In spite of tremendous recent advances in computational gene finding, comprehensive annotation remains a challenge. Peptide mass spectrometry is a powerful tool for researching the dynamic proteome and suggests an attractive approach to discover and validate protein-coding genes. We present algorithms to construct and efficiently search spectra against a genomic database, with no prior knowledge of encoded proteins. By searching a corpus of 18.5 million tandem mass spectra (MS/MS) from human proteomic samples, we validate 39,000 exons and 11,000 introns at the level of translation. We present translation-level evidence for novel or extended exons in 16 genes, confirm translation of 224 hypothetical proteins, and discover or confirm over 40 alternative splicing events. Polymorphisms are efficiently encoded in our database, allowing us to observe variant alleles for 308 coding SNPs. Finally, we demonstrate the use of mass spectrometry to improve automated gene prediction, adding 800 correct exons to our predictions using a simple rescoring strategy. Our results demonstrate that proteomic profiling should play a role in any genome sequencing project.

Algorithms↗

PubMed1996

AltFGF-2, a novel ER-associated FGF-2 protein isoform: its embryonic distribution and functional analysis during neural tube development.

A novel fibroblast growth factor-2 (FGF-2) protein isoform, called altFGF-2, is expressed abundantly during chicken embryogenesis. The amino-terminal domain of the 21.5-kDa altFGF-2 protein diverges completely from the other three FGF-2 proteins due to alternative splicing of their first coding exons. Furthermore, the altFGF-2 protein, in contrast to FGF-2 proteins, is targeted predominantly to the endoplasmic reticulum. In chicken embryos, altFGF-2 and FGF-2 proteins are differentially distributed in several mesodermal structures including developing limbs and kidneys. All four FGF-2 protein isoforms are also expressed in the developing neural tube from early neural plate stages onward. In contrast to FGF-2 proteins, the altFGF-2 isoform is distributed in a dynamic, spatially restricted pattern in notochord and ventral neural tube (floor plate and motor neurons) during specification of neuronal populations. To study the possible shared or differential signaling functions of chicken altFGF-2 and FGF-2 gene products, they were ectopically expressed in the dorsal neural tube aspect of transgenic mouse embryos. Dorsal expression of altFGF-2, but not FGF-2 gene products, induced alteration of neural tube morphology in a significant fraction of mouse embryos (25%). However, no alterations of dorsoventral (d/v) neural tube polarity were detected, indicating that altFGF-2 and FGF-2 gene products either function as permissive cofactors or regulate neural tube growth without affecting establishment of its primary d/v polarity.

Alternative Splicing↗

PubMed2005

Direct sequence comparison of two divergent class I MHC natural killer cell receptor haplotypes.

The murine Ly49 gene family encoding natural killer cell receptors for class I MHC is an example of a rapidly evolving cluster of immune response genes. Determining the genomic sequence of the 129S6/SvEvTac (129S6) Ly49 cluster and comparing it to the known sequence of the C57BL/6 (B6) region provided insight into the mechanisms of Ly49 gene evolution. 129S6 contains 20 Ly49, many of which are pseudogenes and 40% of the genes have no counterpart in the B6 genome. The difference in gene content between these two strains is primarily the result of distinct patterns of gene duplication. Phylogenetic analyses of individual exons showed that Ly49 genes form distinct sub-families and an ancestral haplotype can be surmised. Dotplot analysis supports limited allelism in the two haplotypes; however, large regions of variation punctuate these islands of co-linearity. These variable regions contain a high concentration of repetitive elements that are predicted to contribute to the dynamic evolution of this cluster. The extreme variation in Ly49 haplotype content between mouse strains provides a genetic explanation for the documented differences in natural killer cell phenotype, and also indicates that differences in natural killer cell function observed between B6 and 129-derived gene-targeted mice should be interpreted with caution.

Amino Acid Sequence↗

PubMed2003

Axonal morphogenesis controlled by antagonistic roles of two CRMP subtypes in microtubule organization.

During development, cells undergo dynamic morphological changes by rearrangements of the cytoskeleton including microtubules. However, molecular mechanisms underlying the microtubule remodeling between orientated and disoriented formations are almost unknown. Here we found that novel subtypes of collapsin response mediator proteins (CRMP-As) and the originals (CRMP-Bs), which occur from the alternative usage of different first coding exons, are involved in this conversion of microtubule patterns. Overexpression of CRMP2A and CRMP2B in chick embryonic fibroblasts induced orientated and disoriented patterns of microtubules, respectively. Moreover, sequential overexpression of another subtype overcame the effect of the former expression of the countersubtype. Overexpression experiments in cultured chick retinae showed that CRMP2B promoted axon branching and suppressed axon elongation of ganglion cells, while CRMP2A blocked these effects when co-overexpressed. Our findings suggest that the opposing activities of CRMP2A and CRMP2B contribute to the cellular morphogenesis including neuronal axonogenesis through remodeling of microtubule organization.

Actins↗