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At least 1,099 records · Page 61Linked to original sources
PubMed2000

The pathway for perception and transduction of low-temperature signals in Synechocystis.

Low temperature is an important environmental factor that has effects on all living organisms. Various low-temperature-inducible genes encode products that are essential for acclimation to low temperature, but low-temperature sensors and signal transducers have not been identified. However, systematic disruption of putative genes for histidine kinases and random mutagenesis of almost all the genes in the genome of the cyanobacterium Synechocystis sp. PCC 6803 have allowed us to identify two histidine kinases and a response regulator as components of the pathway for perception and transduction of low-temperature signals. Inactivation, by targeted mutagenesis, of the gene for each of the two histidine kinases and inactivation of the gene for the response regulator depressed the transcription of several lowtemperature-inducible genes.

Bacterial Proteins↗
PubMed2000

Three conserved members of the RNase D family have unique and overlapping functions in the processing of 5S, 5.8S, U4, U5, RNase MRP and RNase P RNAs in yeast.

The biogenesis of a number of RNA species in eukaryotic cells requires 3' processing. To determine the enzymes responsible for these trimming events, we created yeast strains lacking specific 3' to 5' exonucleases. In this work, we describe the analysis of three members of the RNase D family of exonucleases (Rex1p, Rex2p and Rex3p). This work led to three important conclusions. First, each of these exonucleases is required for the processing of distinct RNAs. Specifically, Rex1p, Rex2p and Rex3p are required for 5S rRNA, U4 snRNA and MRP RNA trimming, respectively. Secondly, some 3' exonucleases are redundant with other exonucleases. Specifically, Rex1p and Rex2p function redundantly in 5.8S rRNA maturation, Rex1p, Rex2p and Rex3p are redundant for the processing of U5 snRNA and RNase P RNA, and Rex1p and the exonuclease Rrp6p have an unknown redundant essential function. Thirdly, the demonstration that the Rex proteins can affect reactions that have been attributed previously to the exosome complex indicates that an apparently simple processing step can be surprisingly complex with multiple exonucleases working sequentially in the same pathway.

Conserved Sequence↗
PubMed2002

The mammalian exosome mediates the efficient degradation of mRNAs that contain AU-rich elements.

HeLa cytoplasmic extracts contain both 3'-5' and 5'-3' exonuclease activities that may play important roles in mRNA decay. Using an in vitro RNA deadenylation/decay assay, mRNA decay intermediates were trapped using phosphothioate-modified RNAs. These data indicate that 3'-5' exonucleolytic decay is the major pathway of RNA degradation following deadenylation in HeLa cytoplasmic extracts. Immunodepletion using antibodies specific for the exosomal protein PM-Scl75 demonstrated that the human exosome complex is required for efficient 3'-5' exonucleolytic decay. Furthermore, 3'-5' exonucleolytic decay was stimulated dramatically by AU-rich instability elements (AREs), implicating a role for the exosome in the regulation of mRNA turnover. Finally, PM-Scl75 protein was found to interact specifically with AREs. These data suggest that the interaction between the exosome and AREs plays a key role in regulating the efficiency of ARE-containing mRNA turnover.

Base Composition↗
PubMed2000

Inhibition of mRNA turnover in yeast by an xrn1 mutation enhances the requirement for eIF4E binding to eIF4G and for proper capping of transcripts by Ceg1p.

Null mutants of XRN1, encoding the major cytoplasmic exoribonuclease in yeast, are viable but accumulate decapped, deadenylated transcripts. A screen for mutations synthetic lethal with xrn1Delta identified a mutation in CDC33, encoding eIF4E. This mutation (glutamate to glycine at position 72) affected a highly conserved residue involved in interaction with eIF4G. Synthetic lethality between xrn1 and cdc33 was not relieved by high-copy expression of eIF4G or by disruption of the yeast eIF4E binding protein Caf20p. High-copy expression of a mutant eIF4G defective for eIF4E binding resulted in a dominant negative phenotype in an xrn1 mutant, indicating the importance of this interaction in an xrn1 mutant. Another allele of CDC33, cdc33-1, along with mutations in CEG1, encoding the nuclear guanylyltransferase, were also synthetic lethal with xrn1Delta, whereas mutations in PRT1, encoding a subunit of eIF3, were not. Mutations in CDC33, CEG1, PRT1, PAB1, and TIF4631, encoding eIF4G1, have been shown to lead to destabilization of mRNAs. Although such destabilization in cdc33, ceg1, and pab1 mutants can be partially suppressed by an xrn1 mutation, we observed synthetic lethality between xrn1 and either cdc33 or ceg1 and no suppression of the inviability of a pab1 null mutation by xrn1Delta. Thus, the inhibition of mRNA turnover by blocking Xrn1p function does not suppress the lethality of defects upstream in the turnover pathway but it does enhance the requirement for (7)mG caps and for proper formation of the eIF4E/eIF4G cap recognition complex.

Cell Nucleus↗
PubMed2004

Identification of Edc3p as an enhancer of mRNA decapping in Saccharomyces cerevisiae.

The major pathway of mRNA decay in yeast initiates with deadenylation, followed by mRNA decapping and 5'-3' exonuclease digestion. An in silico approach was used to identify new proteins involved in the mRNA decay pathway. One such protein, Edc3p, was identified as a conserved protein of unknown function having extensive two-hybrid interactions with several proteins involved in mRNA decapping and 5'-3' degradation including Dcp1p, Dcp2p, Dhh1p, Lsm1p, and the 5'-3' exonuclease, Xrn1p. We show that Edc3p can stimulate mRNA decapping of both unstable and stable mRNAs in yeast when the decapping enzyme is compromised by temperature-sensitive alleles of either the DCP1 or the DCP2 genes. In these cases, deletion of EDC3 caused a synergistic mRNA-decapping defect at the permissive temperatures. The edc3Delta had no effect when combined with the lsm1Delta, dhh1Delta, or pat1Delta mutations, which appear to affect an early step in the decapping pathway. This suggests that Edc3p specifically affects the function of the decapping enzyme per se. Consistent with a functional role in decapping, GFP-tagged Edc3p localizes to cytoplasmic foci involved in mRNA decapping referred to as P-bodies. These results identify Edc3p as a new protein involved in the decapping reaction.

Amino Acid Sequence↗
PubMed1999

Nonsense-mediated mRNA decay in health and disease.

All eukaryotes possess the ability to detect and degrade transcripts harboring premature signals for the termination of translation. Despite the ubiquitous nature of nonsense-mediated mRNA decay (NMD) and its demonstrated role in the modulation of phenotypes resulting from selected nonsense alleles, very little is known regarding its basic mechanism or the selective pressure for complete evolutionary conservation of this function. This review will present the current models of NMD that have been generated during the study of model organisms and mammalian cells. The physiological burden of nonsense transcripts and the emerging view that NMD plays a broad and critical role in the regulation of gene expression will also be discussed. Such issues are relevant to the proposal that pharmacological manipulation of NMD will find therapeutic application.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Tissue-specific RNA surveillance? Nonsense-mediated mRNA decay causes collagen X haploinsufficiency in Schmid metaphyseal chondrodysplasia cartilage.

Mutations resulting in a premature termination codon (PTC) are a major cause of inherited disorders, and the majority of these mutant RNA transcripts are subjected to nonsense-mediated mRNA decay (NMD). This RNA surveillance results in reduced mutant allele expression, the extent of which can impact on the clinical severity. The molecular mechanisms of NMD in mammalian cells, its relationship to splicing and translation, downstream sequence elements and binding factors remains only partially understood. Currently there is little information on whether the extent of NMD is gene- or tissue-specific, although nonsense mutation inhibition of RNA splicing has been shown to exhibit some tissue and gene specificity in vitro. Schmid metaphyseal chondrodysplasia results from heterozygous mutations in the gene for collagen X (COL10A1), expressed by the hypertrophic chondrocytes of growth plate cartilage. In one patient a PTC mutation has been shown to result in complete NMD and collagen X haploinsufficiency in cartilage. Here we show that, in this patient, and in another with a different collagen X PTC mutation also leading to complete NMD in cartilage, the mutant mRNAs were not subjected to NMD in non-cartilage cells (lymphoblasts and bone cells). These data suggest that novel RNA surveillance mechanisms may exist in cartilage and that tissue specificity of NMD could be of importance in understanding the molecular pathology of nonsense mutations. Furthermore, the demonstration of collagen X haploinsufficiency in the second patient to be studied at the level of tissue expression, confirms that nonsense mutations leading to complete mutant collagen X mRNA degradation in cartilage is an important molecular cause of SMCD.

Cartilage↗
PubMed2005

Differential nonsense mediated decay of mutated mRNAs in mismatch repair deficient colorectal cancers.

The nonsense-mediated decay (NMD) system normally targets mRNAs with premature termination codons (PTCs) for rapid degradation. We investigated for a putative role of NMD in cancers with microsatellite instability (MSI-H cancers), because numerous mutant mRNAs containing PTC are generated in these tumors as a consequence of their mismatch repair deficiency. Using a quantitative RT-PCR approach in a large series of colorectal cancer cell lines, we demonstrate a significantly increased rate of degradation of mutant mRNAs containing a PTC compared with wild-type. A specific siRNA strategy was used to inhibit RENT-1 and/or RENT-2 activity, two major genes in the NMD system. This allowed us to show that increased degradation of PTC-containing mRNAs in MSI-H tumors was partly dependent upon NMD activity. The efficiency of NMD for the degradation of mutant mRNAs from target genes was highly variable in these cancers. NMD degraded some of them (TGFssRII, MSH3, GRK4), although allowing the persistent expression of others (BAX, TCF-4). This is of particular interest within the context of a proposed conservation of biological activity for the corresponding mutated proteins. We thus propose that NMD might play an important role in the selection of target gene mutations with a functional role in MSI-H carcinogenesis.

Codon, Nonsense↗
PubMed2005

RNA-binding protein is involved in aggregation of light neurofilament protein and is implicated in the pathogenesis of motor neuron degeneration.

Abnormal protein aggregation is emerging as a common theme in the pathogenesis of neurodegenerative disease. Our previous studies have shown that overexpression of untranslated light neurofilament (NF-L) RNA causes motor neuron degeneration in transgenic mice, leads to accumulation of ubiquitinated aggregates in degenerating cultured motor neurons and triggers aggregation of NF-L protein and co-aggregation of mutant SOD1 protein in neuronal cells. Here, we report that p190RhoGEF, an RNA-binding protein that binds to a destabilizing element in NF-L mRNA, is involved in aggregation of NF-L protein and is implicated in the pathogenesis of motor neuron degeneration. We show that p190RhoGEF co-aggregates with unassembled NF-L protein and that co-aggregation is associated with down-regulation of parent NF-L mRNA in neuronal cells. Co-expression of NF-M increases NF assembly and reduces RNA-triggered aggregation as well as loss of solubility of NF-L protein. siRNA-induced down-regulation of p190RhoGEF not only reduces aggregation and promotes assembly of NF-L and NF-M, but also causes reversal of aggregation and recovery of NF assembly in transfected cells. Examination of transgenic models of motor neuron disease shows that prominent aggregates of p190RhoGEF and NF-L and down-regulation of NF-L expression occur in degenerating motor neurons of mice expressing untranslated NF-L RNA or a G93A mutant SOD1 transgene. Moreover, aggregates of p190RhoGEF and NF-L appear as early pathological changes in presymptomatic G93A mutant SOD1 transgenic mice. Together, the findings indicate that p190RhoGEF is involved in aggregation of NF-L protein and support a working hypothesis that aggregation of p190RhoGEF and NF-L is an upstream event triggering neurotoxicity in motor neuron disease.

Animals↗
PubMed2005

Consequences of mutations in the non-coding RMRP RNA in cartilage-hair hypoplasia.

Cartilage-hair hypoplasia (CHH), also known as metaphyseal chondrodysplasia McKusick type (OMIM no. 250250), is an autosomal recessive, multi-systemic disease characterized by disproportionate short stature, fine and sparse hair, deficient cellular immunity and a predisposition to malignancy. It is caused by mutations in RMRP, the RNA component of the ribonucleoprotein complex RNase MRP, and, thus, CHH represents one of few Mendelian disorders caused by mutations in a nuclear encoded, non-coding RNA. While studies in yeast indicate that RMRP contributes to diverse cellular functions, the pathogenesis of the human condition is unknown. Studies of our CHH patient cohort revealed mutations in both the promoter and the transcribed region of RMRP. While mutations in the promoter abolished transcription in vitro, RMRP RNA levels in patients with transcribed mutations were also decreased suggesting an unstable RNA. RMRP mutations introduced into the yeast ortholog, NME1, exhibited normal mitochondrial function, chromosomal segregation and cell cycle progression, while a CHH fibroblast cell line exhibited normal mitochondrial content. However, the most commonly found mutation in CHH patients, 70A>G, caused an alteration in ribosomal processing by altering the ratio of the short versus the long form of the 5.8S rRNA in yeast. Transcriptional profiling of CHH patient RNAs showed upregulation of several cytokines and cell cycle regulatory genes, one of which has been implicated in chondrocyte hypertrophy. These data suggest that alteration of ribosomal processing in CHH is associated with altered cytokine signalling and cell cycle progression in terminally differentiating cells in the lymphocytic and chondrocytic cell lineages.

Animals↗
PubMed1995

1,25(OH)2D3-regulated human keratinocyte proliferation and differentiation: basic studies and their clinical application.

Keratinocytes produce vitamin D, metabolize it to its most biologically active form, 1,25(OH)2D, and respond to the 1,25(OH)2D they produce with a decrease in proliferation and an increase in differentiation. 1,25(OH)2D production by keratinocytes is tightly controlled and changes as the cells differentiate, increasing during the early stages of differentiation and then decreasing again as terminal differentiation ensues. The 1,25(OH)2D produced endogenously or supplied exogenously acts in concert with calcium and products of phosphoinositide metabolism to stimulate the transition from a proliferating basal cell to a terminally differentiated corneocyte. The mechanisms involved include changes in gene transcription and messenger RNA stability. These antiproliferative, prodifferentiating actions of 1,25(OH)2D3 have led to its successful use in psoriasis, a hyperproliferative skin disease, and may lead to its use as a chemopreventive agent in malignancy.

Animals↗
PubMed2002

Selenium influences the turnover of selenocysteine tRNA([Ser]Sec) in Chinese hamster ovary cells.

Selenocysteine transfer RNA (tRNA([Ser]Sec)) is a central molecule in the production of selenium-containing proteins, and may play a role in the regulation of their biosynthesis. Selenium concentration influences both the levels of tRNA([Ser]Sec) and the relative abundance of two isoforms. To study the mechanism by which selenium affects tRNA([Ser]Sec) levels, Chinese hamster ovary (CHO) cells were treated with the transcription inhibitor, actinomycin D, and tRNA([Ser]Sec) levels were determined by Northern blotting, primer extension and reverse-phase column chromatography. Turnover of tRNA([Ser]Sec) in CHO cells was faster than the total tRNA population. Supplementation of the culture media with selenium reduced turnover of tRNA([Ser]Sec), but did not influence turnover of a randomly selected serine tRNA. Inhibition of transcription with actinomycin D resulted in a relative increase in the abundance of the isoform containing methylcarboxymethyl-5'-uridine-2'-O-methylribose in the wobble position of the anticodon. Primer extension studies, which permitted the independent evaluation of the tRNA([Ser]Sec) arising from the introduced mouse gene and that derived from the host CHO gene, indicated an accelerated decline in tRNA([Ser]Sec) derived from both the transfected and the native gene. These results provide additional insight into the levels of regulation that control the translation of selenium containing proteins in mammalian cells.

Animals↗
PubMed2001

Quantitative measurement of transcript levels throughout human preimplantation development: analysis of hypoxanthine phosphoribosyl transferase.

We have developed a competitive reverse transcription-polymerase chain reaction (RT-PCR) sensitive enough to detect and quantify as little as 2-fold differences in gene expression in individual oocytes and embryos throughout human preimplantation development. This RT-PCR assay can be tailored for the examination of any specific gene and so will give a unique insight into human preimplantation development. This technique was used to quantify the level of hypoxanthine phosphoribosyl transferase (HPRT) expression during preimplantation development and to correlate this with embryo sex. The amount of HPRT transcripts present in the unfertilized oocyte was equivalent to 7.7 fg of competitor cDNA. At the 4-cell stage there is a significant drop (P: = 0.0006) to approximately 1.2 fg. There was no detectable difference in the HPRT levels between female and male embryos following 2 days of in-vitro culture. In contrast HPRT gene expression was higher in day 3 female embryos than in males. This is the first study to quantify gene transcripts throughout each stage of human preimplantation development and it indicates that the accumulated HPRT transcripts present in the unfertilized human oocyte undergo extensive destruction following fertilization. This work also suggests that X-inactivation occurs beyond the 8-cell stage of human preimplantation development.

Base Sequence↗
PubMed2003

Quantitative RT-PCR reveals tuberous sclerosis gene, TSC2, mRNA degradation following cryopreservation in the human preimplantation embryo.

The use of cryopreserved human embryos in gene expression studies provides an additional source to the scarce embryos available for research. To validate their use we have implemented a quantitative RT-PCR to characterize the levels of the tuberous sclerosis, TSC2 gene in fresh and frozen-thawed human embryos. Frozen embryos were thawed using two different clinical protocols. In fresh embryos 9.95 fg of TSC2 cDNA was present in the unfertilized oocyte, which was comparable to the level on day 2 of preimplantation development. On day 3 there was a significant drop (P<0.001) to 6.8 fg, followed by an increase in cDNA levels to 10.8 fg (P<0.01) on day 6 at the expanded blastocyst stage. Day 2 frozen embryos possessed 50% less (P<0.001) TSC2 mRNA in comparison to the fresh embryos using thawing protocol one (from frozen to 37 degrees C) and 25% less TSC2 mRNA (P<0.01) with thawing protocol 2 (from frozen to room temperature). After culturing day 2 frozen embryos for an additional day they showed mRNA levels comparable with fresh day 3 embryos. There was no significant difference in the levels of TSC2 mRNA between fresh and frozen day 3 human embryos with either thawing protocol. This study demonstrates that cryopreservation does affect the normal pattern of gene expression during human preimplantation development, and that intact frozen-thawed embryos are not equivalent to their non-frozen counterparts. Furthermore human embryos frozen on day 2 appear to be more susceptible to temperature change than embryos frozen on day 3.

Cellular Senescence↗
PubMed2000

RNA accessibility prediction: a theoretical approach is consistent with experimental studies in cell extracts.

The use of antisense oligodeoxyribonucleotides (ODN) or ribozymes to specifically suppress gene expression is simple in concept and relies on efficient binding of the antisense strand to the target RNA. Although the identification of target sites accessible to base pairing is gradually being overcome by different techniques, it remains a major problem in the antisense and ribozyme approaches. In this study we have investigated the potential of a recent experimental and theoretical approach to predict the local accessibility of murine DNA-methyltransferase (MTase) mRNA in a comparative way. The accessibility of the native target RNA was probed with antisense ODN in cellular extracts. The results strongly correlated with the theoretically predicted target accessibility. This work suggests an effective two-step procedure for predicting RNA accessibility: first, computer-aided selection of ODN binding sites defined by an accessibility score followed by a more detailed experimental procedure to derive information about target accessibility at the single nucleotide level.

Animals↗
PubMed2000

Theoretical design of antisense genes with statistically increased efficacy.

Endogenous expression of antisense RNA represents one major way of applying antisense nucleic acids. To express antisense RNA intracellularly, recombinant antisense genes have to be designed and introduced into cells where the target RNA is encountered. Efficient annealing between the antisense RNA and the target RNA is crucial for efficacy and is strongly influenced by RNA structure. Here we extend structural rules for the design of in vitro transcribed antisense RNAs to the design of recombinant antisense genes. Intracellularly expressed antisense RNA transcripts contain a central antisense portion and additional flanking vector-derived sequences. A computer algorithm was generated to compose large sets of antisense genes, to calculate secondary structures of the transcribed sequences and to select for favorable structures of antisense RNA in terms of annealing and efficacy. The biological test system to measure efficiency of antisense genes was human immunodeficiency virus type 1 (HIV-1) replication in 293T cells. When considering the lower intracellular steady-state levels of favorably structured endogenous transcripts, an antisense effect against HIV-1 replication was observed that was up to 60-fold stronger than that measured for predicted unfavorable species. The computational selection was successful for antisense portions of 300 nt but not 100 nt in length. This theoretical design of antisense genes supports their improved application under time- and labor-saving conditions.

Algorithms↗
PubMed2000

The stress-activated MAP kinase Sty1/Spc1 and a 3'-regulatory element mediate UV-induced expression of the uvi15(+) gene at the post-transcriptional level.

Exposure of Schizosaccharomyces pombe cells to UV light results in increased uvi15(+) gene expression at both the mRNA and protein levels, leading to elevated cell survival. This UV-induced expression of the uvi15(+) gene was reduced in Deltasty1 and Deltawis1 cells lacking the stress-activated protein kinase pathway, but not in DNA damage checkpoint mutants. To further understand the cellular mechanisms responsible for this UV-induced expression, the transcription rate and mRNA half-life were investigated. Transcription run-on assays revealed that the rate of uvi15(+) transcription was increased 1.8-fold regardless of Sty1 when cells were UV irradiated. The half-life of uvi15(+) mRNA was also increased 1.5-fold after UV irradiation, but it was decreased in the Deltasty1 background for both basal and UV-induced mRNAs, indicating that the stress-activated MAPK cascade can mediate UV-induced gene expression by increasing mRNA half-life. Deletion analyses identified a 54 nt element downstream of the distal poly(A) site, which was involved in the increased half-life of uvi15(+) mRNA. These results suggest that both Sty1 and the 3'-regulatory element regulate UV-induced expression of the uvi15(+) gene at the post-transcriptional level.

3' Untranslated Regions↗
PubMed2000

Comparative analysis of secondary structure of insect mitochondrial small subunit ribosomal RNA using maximum weighted matching.

Comparative analysis is the preferred method of inferring RNA secondary structure, but its use requires considerable expertise and manual effort. As the importance of secondary structure for accurate sequence alignment and phylogenetic analysis becomes increasingly realised, the need for secondary structure models for diverse taxonomic groups becomes more pressing. The number of available structures bears little relation to the relative diversity or importance of the different taxonomic groups. Insects, for example, comprise the largest group of animals and yet are very poorly represented in secondary structure databases. This paper explores the utility of maximum weighted matching (MWM) to help automate the process of comparative analysis by inferring secondary structure for insect mitochondrial small subunit (12S) rRNA sequences. By combining information on correlated changes in substitutions and helix dot plots, MWM can rapidly generate plausible models of secondary structure. These models can be further refined using standard comparative techniques. This paper presents a secondary structure model for insect 12S rRNA based on an alignment of 225 insect sequences and an alignment for 16 exemplar insect sequences. This alignment is used as a template for a web server that automatically generates secondary structures for insect sequences.

Algorithms↗