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At least 973 records · Page 54Linked to original sources
PubMed2000

Small angle X-ray scattering reveals a compact intermediate in RNA folding.

We have used small angle X-ray scattering (SAXS) to monitor changes in the overall size and shape of the Tetrahymena ribozyme as it folds. The native ribozyme, formed in the presence of Mg2+, is much more compact and globular than the ensemble of unfolded conformations. Time-resolved measurements show that most of the compaction occurs at least 20-fold faster than the overall folding to the native state, suggesting that a compact intermediate or family of intermediates is formed early and then rearranges in the slow steps that limit the overall folding rate. These results lead to a kinetic folding model in which an initial 'electrostatic collapse' of the RNA is followed by slower rearrangements of elements that are initially mispositioned.

Animals↗
PubMed2000

Gene-expression profile of the ageing brain in mice.

Ageing of the brain leads to impairments in cognitive and motor skills, and is the major risk factor for several common neurological disorders such as Alzheimer disease (AD) and Parkinson disease (PD). Recent studies suggest that normal brain ageing is associated with subtle morphological and functional alterations in specific neuronal circuits, as opposed to large-scale neuronal loss. In fact, ageing of the central nervous system in diverse mammalian species shares many features, such as atrophy of pyramidal neurons, synaptic atrophy, decrease of striatal dopamine receptors, accumulation of fluorescent pigments, cytoskeletal abnormalities, and reactive astrocytes and microglia. To provide the first global analysis of brain ageing at the molecular level, we used oligonucleotide arrays representing 6,347 genes to determine the gene-expression profile of the ageing neocortex and cerebellum in mice. Ageing resulted in a gene-expression profile indicative of an inflammatory response, oxidative stress and reduced neurotrophic support in both brain regions. At the transcriptional level, brain ageing in mice displays parallels with human neurodegenerative disorders. Caloric restriction, which retards the ageing process in mammals, selectively attenuated the age-associated induction of genes encoding inflammatory and stress responses.

Aging↗
PubMed2005

Ultrasound-accelerated formalin fixation of tissue improves morphology, antigen and mRNA preservation.

Formalin fixation and paraffin embedding are conventional tissue preservation and processing methods used for histologic diagnosis in over 90% of cases. However, formalin fixation has three disadvantages: (1) slow fixation (16-24 h) hinders intraoperative decision making, (2) slow quenching of enzymatic activity causes RNA degradation, and (3) extensive molecule modification affects protein antigenicity. Applying high-frequency, high-intensity ultrasound to the formalin fixative cuts fixation time to 5-15 min. Fixation of various tissues such as lymph node, brain, breast, and prostate suggests that, compared to the conventional method, implementation of ultrasound retains superior and more uniform tissue morphology preservation. Less protein antigenicity is altered so that rapid immunohistochemical reactions occur with higher sensitivity and intensity, reducing the need for antigen retrieval pretreatment. Better RNA preservation results in stronger signals in in situ hybridization and longer RNA fragments extracted from fixed tissues, probably due to rapid inhibition of endogenous RNase activity. Molecules extracted from ultrasound-fixed tissues are of greater integrity and quantity compared to conventionally fixed tissues, and thus better support downstream molecular analyses. Overall, ultrasound-facilitated tissue preservation can provide rapid and improved morphological and molecular preservation to better accommodate both traditional and molecular diagnoses.

Antigens↗
PubMed2006

When transcriptome meets metabolome: fast cellular responses of yeast to sudden relief of glucose limitation.

Within the first 5 min after a sudden relief from glucose limitation, Saccharomyces cerevisiae exhibited fast changes of intracellular metabolite levels and a major transcriptional reprogramming. Integration of transcriptome and metabolome data revealed tight relationships between the changes at these two levels. Transcriptome as well as metabolite changes reflected a major investment in two processes: adaptation from fully respiratory to respiro-fermentative metabolism and preparation for growth acceleration. At the metabolite level, a severe drop of the AXP pools directly after glucose addition was not accompanied by any of the other three NXP. To counterbalance this loss, purine biosynthesis and salvage pathways were transcriptionally upregulated in a concerted manner, reflecting a sudden increase of the purine demand. The short-term dynamics of the transcriptome revealed a remarkably fast decrease in the average half-life of downregulated genes. This acceleration of mRNA decay can be interpreted both as an additional nucleotide salvage pathway and an additional level of glucose-induced regulation of gene expression.

Acetic Acid↗
PubMed2002

Modulation of HIV-1 replication by RNA interference.

RNA interference (RNAi) is the process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of messenger RNA in animal and plant cells. In mammalian cells, RNAi can be triggered by 21-nucleotide duplexes of small interfering RNA (siRNA). Here we describe inhibition of early and late steps of HIV-1 replication in human cell lines and primary lymphocytes by siRNAs targeted to various regions of the HIV-1 genome. We demonstrate that synthetic siRNA duplexes or plasmid-derived siRNAs inhibit HIV-1 infection by specifically degrading genomic HIV-1 RNA, thereby preventing formation of viral complementary-DNA intermediates. These results demonstrate the utility of RNAi for modulating the HIV replication cycle and provide evidence that genomic HIV-1 RNA, as it exists within a nucleoprotein reverse-transcription complex, is amenable to siRNA-mediated degradation.

Cell Line↗
PubMed2004

fgf8 mRNA decay establishes a gradient that couples axial elongation to patterning in the vertebrate embryo.

Formation and patterning of the vertebrate embryo occur in a head-to-tail sequence. This progressive mode of body formation from the posterior end of the embryo requires a strict temporal coordination of tissue differentiation--a process involving fibroblast growth factor (FGF) signalling. Here we show that transcription of fgf8 messenger RNA is restricted to the growing posterior tip of the embryo. fgf8 mRNA is progressively degraded in the newly formed tissues, resulting in the formation of an mRNA gradient in the posterior part of the embryo. This fgf8 mRNA gradient is translated into a gradient of FGF8 protein, which correlates with graded phosphorylation of the kinase Akt, a downstream effector of FGF signalling. Such a mechanism provides an efficient means to monitor the timing of FGF signalling, coupling the differentiation of embryonic tissues to the posterior elongation of the embryo. In addition, this mechanism provides a novel model for morphogen gradient formation.

Animals↗
PubMed2004

An eIF4AIII-containing complex required for mRNA localization and nonsense-mediated mRNA decay.

The specification of both the germ line and abdomen in Drosophila depends on the localization of oskar messenger RNA to the posterior of the oocyte. This localization requires several trans-acting factors, including Barentsz and the Mago-Y14 heterodimer, which assemble with oskar mRNA into ribonucleoprotein particles (RNPs) and localize with it at the posterior pole. Although Barentsz localization in the germ line depends on Mago-Y14, no direct interaction between these proteins has been detected. Here, we demonstrate that the translation initiation factor eIF4AIII interacts with Barentsz and is a component of the oskar messenger RNP localization complex. Moreover, eIF4AIII interacts with Mago-Y14 and thus provides a molecular link between Barentsz and the heterodimer. The mammalian Mago (also known as Magoh)-Y14 heterodimer is a component of the exon junction complex. The exon junction complex is deposited on spliced mRNAs and functions in nonsense-mediated mRNA decay (NMD), a surveillance mechanism that degrades mRNAs with premature translation-termination codons. We show that both Barentsz and eIF4AIII are essential for NMD in human cells. Thus, we have identified eIF4AIII and Barentsz as components of a conserved protein complex that is essential for mRNA localization in flies and NMD in mammals.

Active Transport, Cell Nucleus↗
PubMed2004

Nonsense-mediated messenger RNA decay is initiated by endonucleolytic cleavage in Drosophila.

In eukaryotic cells, messenger RNAs harbouring premature termination codons (PTCs) are rapidly degraded by a conserved post-transcriptional mechanism referred to as nonsense-mediated mRNA decay (NMD), which prevents the synthesis of truncated proteins that could be deleterious for the cell. Studies in yeast and mammals indicate that degradation by means of this pathway can occur from both the 5' end of the message (involving decapping and 5'-to-3' exonucleolytic digestion by XRN1) or the 3' end (through accelerated deadenylation and exosome-mediated 3'-to-5' decay). Here we show that, contrary to expectation, degradation of PTC-containing messages in Drosophila is initiated by endonucleolytic cleavage(s) in the vicinity of the nonsense codon. The resulting 5' fragment is rapidly degraded by exonucleolytic digestion by the exosome, whereas the 3' fragment is degraded by XRN1. This decay route is shown for several PTC-containing reporters, as well as an endogenous mRNA that is naturally regulated by NMD. We conclude that, despite conservation in the NMD machinery, PTC-containing transcripts are degraded in Drosophila by a mechanism that differs considerably from those described in yeast and mammals.

Animals↗
PubMed2004

Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs.

RNA interference (RNAi) holds considerable promise as a therapeutic approach to silence disease-causing genes, particularly those that encode so-called 'non-druggable' targets that are not amenable to conventional therapeutics such as small molecules, proteins, or monoclonal antibodies. The main obstacle to achieving in vivo gene silencing by RNAi technologies is delivery. Here we show that chemically modified short interfering RNAs (siRNAs) can silence an endogenous gene encoding apolipoprotein B (apoB) after intravenous injection in mice. Administration of chemically modified siRNAs resulted in silencing of the apoB messenger RNA in liver and jejunum, decreased plasma levels of apoB protein, and reduced total cholesterol. We also show that these siRNAs can silence human apoB in a transgenic mouse model. In our in vivo study, the mechanism of action for the siRNAs was proven to occur through RNAi-mediated mRNA degradation, and we determined that cleavage of the apoB mRNA occurred specifically at the predicted site. These findings demonstrate the therapeutic potential of siRNAs for the treatment of disease.

Animals↗
PubMed2005

A protein interaction network of the malaria parasite Plasmodium falciparum.

Plasmodium falciparum causes the most severe form of malaria and kills up to 2.7 million people annually. Despite the global importance of P. falciparum, the vast majority of its proteins have not been characterized experimentally. Here we identify P. falciparum protein-protein interactions using a high-throughput version of the yeast two-hybrid assay that circumvents the difficulties in expressing P. falciparum proteins in Saccharomyces cerevisiae. From more than 32,000 yeast two-hybrid screens with P. falciparum protein fragments, we identified 2,846 unique interactions, most of which include at least one previously uncharacterized protein. Informatic analyses of network connectivity, coexpression of the genes encoding interacting fragments, and enrichment of specific protein domains or Gene Ontology annotations were used to identify groups of interacting proteins, including one implicated in chromatin modification, transcription, messenger RNA stability and ubiquitination, and another implicated in the invasion of host cells. These data constitute the first extensive description of the protein interaction network for this important human pathogen.

Animals↗
PubMed2006

Endonucleolytic cleavage of eukaryotic mRNAs with stalls in translation elongation.

A fundamental aspect of the biogenesis and function of eukaryotic messenger RNA is the quality control systems that recognize and degrade non-functional mRNAs. Eukaryotic mRNAs where translation termination occurs too soon (nonsense-mediated decay) or fails to occur (non-stop decay) are rapidly degraded. We show that yeast mRNAs with stalls in translation elongation are recognized and targeted for endonucleolytic cleavage, referred to as 'no-go decay'. The cleavage triggered by no-go decay is dependent on translation and involves Dom34p and Hbs1p. Dom34p and Hbs1p are similar to the translation termination factors eRF1 and eRF3 (refs 3, 4), indicating that these proteins might function in recognizing the stalled ribosome and triggering endonucleolytic cleavage. No-go decay provides a mechanism for clearing the cell of stalled translation elongation complexes, which could occur as a result of damaged mRNAs or ribosomes, or as a mechanism of post-transcriptional control.

Cell Cycle Proteins↗
PubMed2006

Mutations in progranulin cause tau-negative frontotemporal dementia linked to chromosome 17.

Frontotemporal dementia (FTD) is the second most common cause of dementia in people under the age of 65 years. A large proportion of FTD patients (35-50%) have a family history of dementia, consistent with a strong genetic component to the disease. In 1998, mutations in the gene encoding the microtubule-associated protein tau (MAPT) were shown to cause familial FTD with parkinsonism linked to chromosome 17q21 (FTDP-17). The neuropathology of patients with defined MAPT mutations is characterized by cytoplasmic neurofibrillary inclusions composed of hyperphosphorylated tau. However, in multiple FTD families with significant evidence for linkage to the same region on chromosome 17q21 (D17S1787-D17S806), mutations in MAPT have not been found and the patients consistently lack tau-immunoreactive inclusion pathology. In contrast, these patients have ubiquitin (ub)-immunoreactive neuronal cytoplasmic inclusions and characteristic lentiform ub-immunoreactive neuronal intranuclear inclusions. Here we demonstrate that in these families, FTD is caused by mutations in progranulin (PGRN) that are likely to create null alleles. PGRN is located 1.7 Mb centromeric of MAPT on chromosome 17q21.31 and encodes a 68.5-kDa secreted growth factor involved in the regulation of multiple processes including development, wound repair and inflammation. PGRN has also been strongly linked to tumorigenesis. Moreover, PGRN expression is increased in activated microglia in many neurodegenerative diseases including Creutzfeldt-Jakob disease, motor neuron disease and Alzheimer's disease. Our results identify mutations in PGRN as a cause of neurodegenerative disease and indicate the importance of PGRN function for neuronal survival.

Cell Survival↗
PubMed2006

An RNA gene expressed during cortical development evolved rapidly in humans.

The developmental and evolutionary mechanisms behind the emergence of human-specific brain features remain largely unknown. However, the recent ability to compare our genome to that of our closest relative, the chimpanzee, provides new avenues to link genetic and phenotypic changes in the evolution of the human brain. We devised a ranking of regions in the human genome that show significant evolutionary acceleration. Here we report that the most dramatic of these 'human accelerated regions', HAR1, is part of a novel RNA gene (HAR1F) that is expressed specifically in Cajal-Retzius neurons in the developing human neocortex from 7 to 19 gestational weeks, a crucial period for cortical neuron specification and migration. HAR1F is co-expressed with reelin, a product of Cajal-Retzius neurons that is of fundamental importance in specifying the six-layer structure of the human cortex. HAR1 and the other human accelerated regions provide new candidates in the search for uniquely human biology.

Aging↗
PubMed2004

From mRNA to tumor suppressor.

The combination of inhibition of RNA degradation and comparative genomic scanning is a powerful new method for detecting gene disruptions. The utility of the method is well-illustrated by a series of observations linking the ephrin receptor EPHB2 to prostate cancer.

Emetine↗
PubMed2004

Nonsense surveillance regulates expression of diverse classes of mammalian transcripts and mutes genomic noise.

Premature termination codons induce rapid transcript degradation in eukaryotic cells through nonsense-mediated mRNA decay (NMD). This pathway can modulate phenotypes arising from nonsense or frameshift mutations, but little is known about the physiologic role of NMD in higher eukaryotes. To address this issue, we examined expression profiles in mammalian cells depleted of Rent1 (also called hUpf1), a factor essential for NMD. Upregulated transcripts included those with upstream open reading frames in the 5' untranslated region, alternative splicing that introduces nonsense codons or frameshifts, introns in the 3' untranslated region or selenocysteine codons. Transcripts derived from ancient transposons and endogenous retroviruses were also upregulated. These RNAs are unified by the presence of a spliced intron at least 50 nucleotides downstream of a termination codon, a context sufficient to initiate NMD. Consistent with direct regulation by NMD, representative upregulated transcripts decayed more slowly in cells deficient in NMD. In addition, inhibition of NMD induced by amino acid starvation upregulated transcripts that promote amino acid homeostasis. These results document that nonsense surveillance is a crucial post-transcriptional regulatory event that influences the expression of broad classes of physiologic transcripts, has been functionally incorporated into essential homeostatic mechanisms and suppresses expression of evolutionary remnants.

Amino Acids↗