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At least 1,135 records · Page 63Linked to original sources
PubMed2006

'Evidence of an auxin signal pathway, microRNA167-ARF8-GH3, and its response to exogenous auxin in cultured rice cells'.

MicroRNA167 (miR167) was shown to cleave auxin responsive factor 8 (ARF8) mRNA in cultured rice cells. MiR167 level was found to be controlled by the presence of auxin in the growth medium. When cells grew in auxin-free medium, miR167 level decreased, resulting in an increase in the level of ARF8 mRNA. Cells growing in the normal growth medium containing auxin showed a reversed trend. It was also shown that expression of OsGH3-2, an rice IAA-conjugating enzyme, was positively regulated by ARF8. Delivery of synthesized miR167 into cells led to decrease of both ARF8 mRNA and OsGH3-2 mRNA. This study provides an evidence in which the exogeneous auxin signal is transduced to OsGH3-2 through miR167 and ARF8 in sequence. This proposed auxin signal transduction pathway, auxin-miR167-ARF8-OsGH3-2, could be, in conjunction with the other microRNA-mediated auxin signals, an important one for responding to exogeneous auxin and for determining the cellular free auxin level which guides appropriate auxin responses.

Cells, Cultured↗
PubMed2005

Trans insertion-splicing: ribozyme-catalyzed insertion of targeted sequences into RNAs.

A group I intron-derived ribozyme from Pneumocystis carinii has been previously shown to bind an exogenous RNA substrate, splice out an internal segment, and then ligate the two ends back together (the trans excision-splicing reaction). We demonstrate that this same ribozyme can perform a trans insertion-splicing (TIS) reaction, where the ribozyme binds two exogenous RNA substrates and inserts one directly into the other. Reactions were optimized for both yield and rate, with optimum reactions carried out in 10 mM MgCl(2) for 2 h. Reaction products are stable, with no visible loss at extended times. The ribozyme recognizes the two substrates primarily through base pairing and requires an omegaG on the ribozyme and an omegaG on the sequence being inserted. We give evidence that the reaction mechanism is not the reverse of the trans excision-splicing reaction, but is composed of three steps, with intermediates attached to the ribozyme. Surprisingly, the internal guide sequence of the ribozyme is utilized to sequentially bind both substrates, forming independent P1 helices. This is an indication that ribozymes with essentially the native intron sequence can catalyze reactions significantly more dynamic and complex than self-splicing. The implications of group I intron-derived ribozymes being able to catalyze this unique reaction, and via this mechanism, are discussed.

Catalysis↗
PubMed2004

Establishing mathematical laws of genomic variation.

As the biological arm of the Rasch community, genomic measurement is concerned with asserting and testing hypotheses regarding the quantitative status of genomic variables, including alleles, genotypes, gene expression levels, and phenotypes, as well as DNA, RNA, and protein sequence information. The defining goal of this scientific paradigm, in contrast to the sample-dependent model-fitting and deterministic hypothesis testing of classical statistical genetics, is the identification, validation, and maintenance of a common unit of genomic measurement that maintains its magnitude and meaning, within an allowable range of error, regardless of the laboratory technology used to generate outcomes or the particular group of individuals or organisms under investigation. Such an invariant metric, the basis of a standard genometric scale and associated system of genomic metrology, can be identified, validated, and maintained through 1) routine implementation of the Rasch family of measurement models to construct sample- and scale-free measures from different types of genomic data and 2) cross-calibration of genomic measurement instruments between and among researchers, laboratories, universities, corporations, and databases. This manuscript provides an introductory overview of the guiding principles of fundamental measurement theory and the work of Rasch, connects these concepts to well-known tenets of population genetics, and highlights the potential benefits, both theoretical and applied, associated with achieving objectivity in genomic measurement.

Animals↗
PubMed1997

Intron-dependent enzymatic formation of modified nucleosides in eukaryotic tRNAs: a review.

In eukaryotic cells, especially in yeast, several genes encoding tRNAs contain introns. These are removed from pre-tRNAs during the maturation process by a tRNA-specific splicing machinery that is located within the nucleus at the nuclear envelope. Before and after the intron removal, several nucleoside modifications are added in a stepwise manner, but most of them are introduced prior to intron removal. Some of these early nucleoside modifications are catalyzed by intron-dependent enzymes while most of the others are catalyzed in an intron-independent manner. In the present paper, we review all known cases where the nucleoside modifications were shown to depend strictly on the presence of an intron. These are pseudouridines at anticodon positions 34, 35 and 36 and 5-methylcytosine at position 34 of several eukaryotic tRNAs. One common property of the corresponding intron-dependent modifying enzymes is that their activities are essentially dependent on the local specific architecture of the pre-tRNA molecule that comprises the anticodon stem and loop prolonged by the intron domain. Thus introns clearly serve as internal (cis-type) RNAs that guide nucleoside modifications by providing transient target sites in tRNA for selected nuclear modifying enzymes. This situation may be similar to the recently discovered (trans-type) snoRNA-guided process of ribose methylations of ribosomal RNAs within the nucleolus of eukaryotic cells.

Animals↗
PubMed1998

Sequence and structural elements of methylation guide snoRNAs essential for site-specific ribose methylation of pre-rRNA.

Site-specific 2'-O-ribose methylation of eukaryotic rRNAs is guided by small nucleolar RNAs (snoRNAs). The methylation guide snoRNAs carry long perfect complementaries to rRNAs. These antisense elements are located either in the 5' half or in the 3' end region of the snoRNA, and are followed by the conserved D' or D box motifs, respectively. An uninterrupted helix formed between the rRNA and the antisense element of the snoRNA, in conjunction with the adjacent D' or D box, constitute the recognition signal for the putative methyltransferase. Here, we have identified an additional essential box element common to methylation guide snoRNAs, termed the C' box. We show that the C' box functions in concert with the D' box and plays a crucial role in the methyltransfer reaction directed by the upstream antisense element and the D' box. We also show that an internal fragment of U24 methylation guide snoRNA, encompassing the upstream antisense element and the D' and C' box motifs, can support the site-specific methylation of rRNA. This strongly suggests that the C box of methylation guide snoRNAs plays an essential role in the methyltransfer reaction guided by the 3'-terminal antisense element and the D box of the snoRNA.

Base Sequence↗
PubMed2006

Structure-function analysis of rotavirus NSP2 octamer by using a novel complementation system.

Viral inclusion bodies, or viroplasms, that form in rotavirus-infected cells direct replication and packaging of the segmented double-stranded RNA (dsRNA) genome. NSP2, one of two rotavirus proteins needed for viroplasm assembly, possesses NTPase, RNA-binding, and helix-unwinding activities. NSP2 of the rotavirus group causing endemic infantile diarrhea (group A) was shown to self-assemble into large doughnut-shaped octamers with circumferential grooves and deep clefts containing nucleotide-binding histidine triad (HIT)-like motifs. Here, we demonstrate that NSP2 of group C rotavirus, a group that fails to reassort with group A viruses, retains the unique architecture of the group A octamer but differs in surface charge distribution. By using an NSP2-dependent complementation system, we show that the HIT-dependent NTPase activity of NSP2 is necessary for dsRNA synthesis, but not for viroplasm formation. The complementation system also showed that despite the retention of the octamer structure and the HIT-like fold, group C NSP2 failed to rescue replication and viroplasm formation in NSP2-deficient cells infected with group A rotavirus. The distinct differences in the surface charges on the Bristol and SA11 NSP2 octamers suggest that charge complementarity of the viroplasm-forming proteins guides the specificity of viroplasm formation and, possibly, reassortment restriction between rotavirus groups.

Amino Acid Sequence↗
PubMed2002

Statistical mechanics of secondary structures formed by random RNA sequences.

The formation of secondary structures by a random RNA sequence is studied as a model system for the sequence-structure problem omnipresent in biopolymers. Several toy energy models are introduced to allow detailed analytical and numerical studies. First, a two-replica calculation is performed. By mapping the two-replica problem to the denaturation of a single homogeneous RNA molecule in six-dimensional embedding space, we show that sequence disorder is perturbatively irrelevant, i.e., an RNA molecule with weak sequence disorder is in a molten phase where many secondary structures with comparable total energy coexist. A numerical study of various models at high temperature reproduces behaviors characteristic of the molten phase. On the other hand, a scaling argument based on the external statistics of rare regions can be constructed to show that the low-temperature phase is unstable to sequence disorder. We performed a detailed numerical study of the low-temperature phase using the droplet theory as a guide, and characterized the statistics of large-scale, low-energy excitations of the secondary structures from the ground state structure. We find the excitation energy to grow very slowly (i.e., logarithmically) with the length scale of the excitation, suggesting the existence of a marginal glass phase. The transition between the low-temperature glass phase and the high-temperature molten phase is also characterized numerically. It is revealed by a change in the coefficient of the logarithmic excitation energy, from being disorder dominated to being entropy dominated.

Biophysics↗
PubMed2003

Expression profiling via novel multiplex assay allows rapid assessment of gene regulation in defined signalling pathways.

The current interest in expression of groups of functionally related genes creates a demand for novel experimental tools. We describe a multiplex ligation-dependent amplification procedure (RT-MLPA), which accurately quantifies up to 45 transcripts of interest in a one-tube assay. The output, a set of fluorescent DNA fragments, is analysed via capillary sequencer and spreadsheet software. The procedure is highly sensitive and reproducible over a 100-fold range of input RNA, with excellent compatibility with RT-PCR and microarrays. We targeted two comprehensive sets of human genes: 35 apoptosis regulators and 30 genes involved in inflammation. Both probe sets accurately assessed specific changes in gene expression in two relevant model systems. Stimulation of lymphocytes with various Toll-like receptor (TLR) ligands induced distinct inflammatory profiles. Furthermore, osteosarcoma cells treated with cytostatic drugs showed as primary response strong up-regulation of the apoptogenic p53-inducible PUMA transcript. Suppression by RNAi validated that indeed Puma expression was responsible for apoptosis induction. Thus, RT-MLPA enables relevant changes in transcription patterns to be quickly pinpointed and guide further experiments. This can be an advantage compared to hypothesis-free whole genome screens where large numbers of differentially expressed genes can obscure functional interpretation.

Apoptosis↗
PubMed2004

A protein interaction framework for human mRNA degradation.

The degradation of mRNA is an important regulatory step in the control of gene expression. However, mammalian RNA decay pathways remain poorly characterized. To provide a framework for studying mammalian RNA decay, a two-hybrid protein interaction map was generated using 54 constructs from 38 human proteins predicted to function in mRNA decay. The results provide evidence for interactions between many different proteins required for mRNA decay. Of particular interest are interactions between the poly(A) ribonuclease and the exosome and between the Lsm complex, decapping factors, and 5'-->3' exonucleases. Moreover, multiple interactions connect 5'-->3' and 3'-->5' decay proteins to each other and to nonsense-mediated decay factors, providing the opportunity for coordination between decay pathways. The interaction network also predicts the internal organization of the exosome and Lsm complexes. Additional interactions connect mRNA decay factors to many novel proteins and to proteins required for other steps in gene expression. These results provide an experimental insight into the organization of proteins required for mRNA decay and their coupling to other cellular processes, and the physiological relevance of many of these interactions are supported by their evolutionary conservation. The interactions also provide a wealth of hypotheses to guide future research on mRNA degradation and demonstrate the power of exhaustive protein interaction mapping in aiding understanding of uncharacterized protein complexes and pathways.

Carrier Proteins↗
PubMed2024

PTGS is dispensable for the initiation of epigenetic silencing of an active transposon in Arabidopsis.

Transposable elements (TEs) are repressed in plants through transcriptional gene silencing (TGS), maintained epigenetic silencing marks such as DNA methylation. However, the mechanisms by which silencing is first installed remain poorly understood in plants. Small interfering (si)RNAs and post-transcriptional gene silencing (PTGS) are believed to mediate the initiation of TGS by guiding the first deposition of DNA methylation. To determine how this silencing installation works, we took advantage of ÉVADÉ (EVD), an endogenous retroelement in Arabidopsis, able to recapitulate true de novo silencing with a sequence of PTGS followed by a TGS. To test whether PTGS is required for TGS, we introduce active EVD into RNA-DEPENDENT-RNA-POLYMERASE-6 (RDR6) mutants, an essential PTGS component. EVD activity and silencing are monitored across several generations. In the absence of PTGS, silencing of EVD is still achieved through installation of RNA-directed DNA methylation (RdDM). Our study shows that PTGS is dispensable for de novo EVD silencing. Although we cannot rule out that PTGS might facilitate TGS, or control TE activity, initiation of epigenetic silencing can take place in its absence.

Arabidopsis↗
PubMed2004

DNMT3L stimulates the DNA methylation activity of Dnmt3a and Dnmt3b through a direct interaction.

In mammals, the resetting of DNA methylation patterns in early embryos and germ cells is crucial for development. Two DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns. Dnmt3L, a member of the Dnmt3 family, has been reported to be necessary for maternal methylation imprinting, possibly by interacting with Dnmt3a and/or Dnmt3b (Hata, K., Okano, M., Lei, H., and Li, E. (2002) Development 129, 1983-1993). In the present study, the effect of DNMT3L, a human homologue of Dnmt3L, on the DNA methylation activity of mouse Dnmt3a and Dnmt3b was examined in vitro. DNMT3L enhanced the DNA methylation activity of Dnmt3a and Dnmt3b about 1.5-3-fold in a dose-dependent manner but did not enhance the DNA methylation activity of Dnmt1. Although the extents of stimulation were different, a stimulatory effect on the DNA methylation activity was observed for all of the substrate DNA sequences examined, such as those of the maternally methylated SNRPN and Lit-1 imprinting genes, the paternally methylated H19 imprinting gene, the CpG island of the myoD gene, the 5 S ribosomal RNA gene, an artificial 28-bp DNA, poly(dG-dC)-poly(dG-dC), and poly(dI-dC)-poly(dI-dC). DNMT3L could not bind to DNA but could bind to Dnmt3a and Dnmt3b, indicating that the stimulatory effect of DNMT3L on the DNA methylation activity may not be due to the guiding of Dnmt3a and Dnmt3b to the targeting DNA sequence but may comprise a direct effect on their catalytic activity. The carboxyl-terminal half of DNMT3L was found to be responsible for the enhancement of the enzyme activity.

Animals↗
PubMed2026

Lactylation-related immune-metabolic dysregulation defines prognostic and therapeutic stratification in lung adenocarcinoma.

BACKGROUND: Lactylation links lactate metabolism with inflammatory signaling and immune regulation in tumors. However, its cellular distribution and translational value in lung adenocarcinoma (LUAD) remain unclear. METHODS: Single-cell RNA-sequencing datasets GSE189357 and GSE171145 were integrated to characterize lactylation-related activity, intercellular communication, and malignant epithelial cell states in LUAD. Single-cell-derived lactylation-related differentially expressed genes were mapped to TCGA-LUAD and multiple GEO cohorts. Univariate Cox regression and machine learning algorithms were used to construct a lactylation-related prognostic signature (LRPS). The associations of LRPS with prognosis, immunotherapy response, drug sensitivity, genomic alterations, immune infiltration, and inflammation- and metabolism-related pathways were evaluated. KRT7 was further validated using virtual knockout analysis, spatial transcriptomics, and in vitro and in vivo experiments. RESULTS: lactylation-related transcriptional activity showed heterogeneous distribution across LUAD cell populations and was associated with altered cell-cell communication. In malignant epithelial cells, LRTS-high and LRTS-low states exhibited distinct metabolic, inflammatory, and tumor-related pathway activities. LRPS showed stable prognostic performance in TCGA-LUAD and multiple GEO cohorts and remained an independent prognostic factor. Low LRPS was associated with greater potential benefit from immunotherapy, whereas different LRPS groups displayed distinct drug sensitivity, genomic alteration, and immune microenvironment patterns. KRT7 was highly expressed in LUAD and associated with poor prognosis. KRT7 knockdown suppressed LUAD cell proliferation, migration, invasion, colony formation, and tumor growth in vivo. CONCLUSIONS: This study identifies lactylation-related immune-metabolic dysregulation as a clinically relevant feature of LUAD and develops a single-cell-guided LRPS for prognosis and therapeutic stratification. KRT7 emerged as an LRPS-related functional candidate with experimentally supported roles in malignant LUAD phenotypes.

Immunotherapy↗
PubMed1993

Second messenger regulation of mRNA for corticotropin-releasing factor.

An understanding of how second messengers and their ligands are coupled to CRF gene activation is necessary if we are to understand the regulation of the CRF gene in physiological and pathological states. The protein kinase A, protein kinase C and glucocorticoid second messenger systems mediate most of the regulation of the CRF gene. In in vitro systems, CRF gene expression is stimulated 20-30-fold by activation of either the protein kinase A or the protein kinase C system. Glucocorticoid is able to inhibit stimulation via both pathways, but appears to be more effective in repressing activation mediated by protein kinase C. Glucocorticoid negative regulation requires the presence of glucocorticoid receptor possessing an intact DNA-binding domain, suggesting that this effect involves binding of the receptor to the CRF promoter. These in vitro studies should serve to guide investigators towards the possible mechanisms underlying CRF gene regulation in vivo.

Animals↗
PubMed2005

Schwann cell-autonomous role of neuropilin-2.

Neuropilins and group A plexins are components of receptor complexes for class 3 semaphorins, gradients of which help to guide migration of neural progenitor cells and axonal growth cones during development. We demonstrated previously that neuropilins and class 3 semaphorins are induced in sciatic nerve by crush or transection. We now report that in cultured rat Schwann cells, expression of mRNA encoding neuropilin-2 (NRP2) and plexin-A3 (PlexA3), proteins involved in semaphorin-3F (Sema3F) signal transduction, is diminished markedly by forskolin, an adenylate cyclase activator that, like axonal contact, induces Schwann cell synthesis of myelin lipids and proteins. Interestingly, Schwann cell expression of mRNA encoding NRP1, which participates in Sema3A signaling, is not downregulated by forskolin. Antibodies that recognize ectodomains of NRP2 but not control antibodies prevented cultured Schwann cells from aligning in parallel and forming columns. These results are consistent with the view that in nerves undergoing Wallerian degeneration, Schwann cell NRP2 facilitates assembly of Schwann cells into the tubular aggregates (bands of Büngner) that guide regenerating axons.

Animals↗
PubMed1992

Differential activation of NGF receptor and early response genes in neural crest-derived cells.

Nerve growth factor (NGF) binds to a specific cell surface receptor (NGFR) that exists in high affinity (now called trk) and low affinity (now called p75NGFR) forms. NGF-responsive neurons express both forms of the receptor, while Schwann cells, during early development and after nerve injury, express only low affinity p75NGFR. In an attempt to determine whether NGF alters patterns of gene expression in p75NGFR-bearing Schwann cells, we examined the regulation of three early response genes (NGFI-A, NGFI-B, and c-fos) in JS1 rat schwannoma cells. Although these genes are markedly activated by NGF in PC12 (rat pheochromocytoma) cells, NGF has no effect on their transcription in JS1 cells. In contrast to PC12 cells, NGFI-A and NGFI-B are constitutively expressed in JS1 cells, whereas the c-fos gene is not expressed. Treating JS1 cells with cycloheximide (CHX), an inhibitor of protein synthesis that commonly potentiates induction of early response genes by presumably inhibiting synthesis of transcriptional repressors, markedly induces the transcription of NGFI-A and c-fos as well as p75NGFR genes. These data suggest that transcriptional repression plays a major role in the regulation of these genes and that the markedly different regulation of NGFI-A, NGFI-B, and c-fos, all of which encode transcriptional regulators, may be important in guiding the differentiation of these cell types.

Animals↗
PubMed2005

Prospects for personalized cardiovascular medicine: the impact of genomics.

Sequencing of the human genome has ushered in prospects for individualizing cardiovascular health care. There is growing evidence that the practice of cardiovascular medicine might soon have a new toolbox to predict and treat disease more effectively. The Human Genome Project has spawned several important "omic" technologies that allow "whole genome" interrogation of sequence variation (re-sequencing, genotyping, comparative genome hybridization), transcription (expression profiling, tissue arrays), proteins (gas or liquid chromatography and tandem mass spectroscopy [MS]), and metabolites (MS or nuclear magnetic resonance profiling); deoxyribonucleic acid, ribonucleic acid, protein, and metabolic approaches all provide more exacting detail of cardiovascular disease mechanisms and, in some cases, are redefining its taxonomy. Pharmacogenomic approaches are emerging across broad classes of cardiovascular therapeutics to assist practitioners in making more precise decisions about which drugs to give to which patients to optimize the benefit-to-risk ratio. Molecular imaging is developing chemical and biological probes that can sense molecular pathway mechanisms that will allow us to monitor health and disease. Together, these tools will enable a paradigm shift from genetic medicine--on the basis of the study of individual inherited characteristics, most often single genes--to genomic medicine, which by its nature is comprehensive and focuses on the functions and interactions of multiple genes and gene products, among themselves and with their environment. The information gained from such analyses, in combination with clinical data, is now allowing us to assess individual risks and guide clinical management and decision-making, all of which form the basis for cardiovascular genomic medicine.

Cardiology↗
PubMed2005

Subnuclear organelles: new insights into form and function.

The cell nucleus is a complex and highly dynamic environment with many functionally specialized regions of substructure that form and maintain themselves in the absence of membranes. Relatively little is known about the basic physical properties of the nuclear interior or how domains within the nucleus are structurally and functionally organized and interrelated. Here, we summarize recent data that shed light on the structural and functional properties of three prominent subnuclear organelles--nucleoli, Cajal bodies (CBs) and speckles. We discuss how these findings impact our understanding of the guiding principles of nuclear organization and various types of human disease.

Animals↗
PubMed1993

Interlocked circle formation by group I introns: structural requirements and mechanism.

Precursor RNA transcribed from the yeast mitochondrial gene coding for the large ribosomal RNA contains a group I intron that can excise itself in vitro. Apart from group I specific sequence elements the intron also contains a gene encoding a DNA endonuclease involved in intron dispersal. A precursor RNA derivative from which this gene has been removed self-splices efficiently, but due to activation of cryptic opening sites located in the 5' exon, the 3' part of this exon is sometimes co-excised with the intron. Upon further reaction, this enlarged intron molecules give rise to interlocked circles, comprising small circles derived from 5' exon parts and large circles of the intron. Sequence comparison between cryptic opening sites and authentic splice sites reveals in most cases homology with the 3' exon part that is capable of interacting with the Internal Guide Sequence. The role of the IGS was further substantiated by replacing the cryptic opening sites with well defined sequences of authentic splice sites: one corresponding to the 3' splice site and its mutant derivatives, the other to a fragment containing the natural 5'-3' exon junction. Precursor RNAs derived from these constructs give rise to interlocked circles, and mutation studies confirm that the 3' exon nucleotides flanking a 3' splice site are essential for their formation. The results underline the crucial role of the IGS in interlocked circle formation which behaves similarly as in the normal self-splicing reactions. It has been proposed that the two short helices formed by basepairing of the IGS with the 5' and 3' exon can co-axially stack on top of each other forming a quasi continuous RNA double helix or pseudoknot. We present a model explaining how transesterification reactions of a mutant precursor RNA in such a pseudoknot can lead to interlocked circles. The experiments support the notion that a similar structure is also operative in splicing of wild type precursor RNA.

Base Sequence↗