Protocol for small-scale RNA isolation and transcriptional profiling of developing Arabidopsis seeds.
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Arabidopsis↗
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Recycling of a regulatory protein by degradation of the RNA to which it binds.
When Bacillus subtilis is grown in the presence of excess tryptophan, transcription of the trp operon is regulated by binding of tryptophan-activated TRAP to trp leader RNA, which promotes transcription termination in the trp leader region. Transcriptome analysis of a B. subtilis strain lacking polynucleotide phosphorylase (PNPase; a 3'-to-5' exoribonuclease) revealed a striking overexpression of trp operon structural genes when the strain was grown in the presence of abundant tryptophan. Analysis of trp leader RNA in the PNPase(-) strain showed accumulation of a stable, TRAP-protected fragment of trp leader RNA. Loss of trp operon transcriptional regulation in the PNPase(-) strain was due to the inability of ribonucleases other than PNPase to degrade TRAP-bound leader RNA, resulting in the sequestration of limiting TRAP. Thus, in the case of the B. subtilis trp operon, specific ribonuclease degradation of RNA in an RNA-protein complex is required for recycling of an RNA-binding protein. Such a mechanism may be relevant to other systems in which limiting concentrations of an RNA-binding protein must keep pace with ongoing transcription.
Bacillus subtilis↗
Identification of a novel latency-specific splice donor signal within the herpes simplex virus type 1 2.0-kilobase latency-associated transcript (LAT): translation inhibition of LAT open reading frames by the intron within the 2.0-kilobase LAT.
Herpes simplex virus type 1 establishes latent infection in trigeminal ganglia of mice infected via the eye. A family of three colinear viral transcripts (LATs), 2.0, 1.5, and 1.45 kb, is present in latently infected ganglia. To characterize these LATs, lambda gt10 cDNA libraries were constructed with RNAs isolated from the trigeminal ganglia of latently infected mice. A series of recombinant bacteriophage were isolated containing cDNA inserts covering 1.7 kb of the 2.0-kb LAT. Splice junctions of the smaller LATs and the 3' end of the 2.0-kb LAT were identified by sequence analysis of RNA polymerase chain reaction products. No splice acceptor site, which does not support the hypotheses that the 2.0-kb LAT is an intron. However, the data are consistent with the possibility of a short leader sequence or multiple LAT transcription start sites. To generate the smaller 1.5- and 1.45-kb LATs, there is a 559-nucleotide intron spliced from the 2.0-kb LAT in strain F and a 556-nucleotide intron in strain 17+. The nucleotide sequences at the 5' and 3' ends of these introns are characteristic of spliced transcripts from eukaryotic protein-encoding genes, with one significant difference; i.e., the 5' end of the LAT intron is GC instead of the consensus sequence GT. This splice donor sequence is conserved in herpes simplex virus type 1 strains F, 17+, and KOS. Processing of the 2.0-kb LAT to form the spliced LATs preserves two open reading frames (ORFs) at the 3' end of the LATs; no new ORFs are created. Splicing of the LATs positions a 276-nucleotide leader sequence close to these ORFs and removes an intron that inhibits their translation in vitro. The novel 5' structure of the intron within the 2.0-kb LAT may be part of a control mechanism for transcription processing that results in splicing of the LATs only in sensory neurons during latent infection and reactivation but not during the viral replication cycle.
Animals↗
Universal structural features of prokaryotic and eukaryotic ribosomal 5S RNA derived from comparative analysis of their sequences.
An extensive comparative analysis of more than fifty available sequences of ribosomal 5S RNA has been made. Both for prokaryotic and eukaryotic 5S RNA a generalized secondary structure is presented which is similar to that suggested by Nishikawa and Takemura modified in few positions only. Both generalized secondary structures contain five main helical regions and a high base-pairing content of about 65 +/- 5%. The general structural architecture of prokaryotic and eukaryotic 5S RNA molecules appears to be very similar with minor modifications within particular subgroups of organisms. Conserved and semiconserved nucleotides are accumulated in the single stranded parts of 5S RNA. Functional importance was suggested for some of these regions; other short conserved nucleotide stretches may be involved in the folding of 5S RNA molecules. In particular, we propose a tertiary base-pairing interaction between the universal invariant GUA sequence (positions 76-78 and 75-77 in prokaryotic and eukaryotic 5S RNA, respectively) and the complementary conserved CPuU sequence (positions 38-40 and 36-38) in a parallel manner. A molecular model of the 5S RNA of human KB cells was constructed, which verifies the proposed tertiary interaction, probably stabilizing the two neighboured helices E and D and a stacking arrangement of the bases in the sequence positions 67-108 (and 70-106) in eukaryotic (and prokaryotic) 5S RNAs, respectively.
Animals↗
Distinct and complementary information provided by use of tissue and DNA microarrays in the study of breast tumor markers.
Emerging high-throughput screening technologies are rapidly providing opportunities to identify new diagnostic and prognostic markers and new therapeutic targets in human cancer. Currently, cDNA arrays allow the quantitative measurement of thousands of mRNA expression levels simultaneously. Validation of this tool in hospital settings can be done on large series of archival paraffin-embedded tumor samples using the new technique of tissue microarray. On a series of 55 clinically and pathologically homogeneous breast tumors, we compared for 15 molecules with a proven or suspected role in breast cancer, the mRNA expression levels measured by cDNA array analysis with protein expression levels obtained using tumor tissue microarrays. The validity of cDNA array and tissue microarray data were first verified by comparison with quantitative reverse transcriptase-polymerase chain reaction measurements and immunohistochemistry on full tissue sections, respectively. We found a good correlation between cDNA and tissue array analyses in one-third of the 15 molecules, and no correlation in the remaining two-thirds. Furthermore, protein but not RNA levels may have prognostic value; this was the case for MUC1 protein, which was studied further using a tissue microarray containing approximately 600 tumor samples. For THBS1 the opposite was observed because only RNA levels had prognostic value. Thus, differences extended to clinical prognostic information obtained by the two methods underlining their complementarity and the need for a global molecular analysis of tumors at both the RNA and protein levels.
Base Sequence↗
Gene expression profiling of the bone marrow mononuclear cells from patients with myelodysplastic syndrome.
The gene expression pattern of bone marrow mononuclear cells (BMNCs) from 10 patients with myelodysplastic syndrome (MDS) was studied by two-color cDNA microarray techniques. To confirm the microarray results, a semiquantitative RT-PCR was performed to analyze gene expression in fifty additional MDS patients. Ninety-five genes were shown to be abnormally expressed in at least five MDS patients compared to normal controls, involving cell growth and differentiation regulation, cell cycle control, signaling and redox; such as thrombospondin 1, phosphatase and tensin homolog, MAD, DNA-damage-inducible transcript 3 (DDIT3), ets variant gene 1 (ETV1), and G1 to S phase transition 1. CD36 was also revealed up-regulated in 4 cases. MDS patients in early and advanced stages could be clustered into two distinct groups by hierarchical clustering, wherein a case with isolated thrombocytopenia and other RA patients were clustered into two subgroups. Consistent expression patterns of 3/5 (60%) genes were confirmed by semiquantitative RT-PCR. Further analysis showed the different transcript levels of RNAHP, DDIT3 in patients with MDS in different stages, AML, and normal controls. Meanwhile, the different significance of RNAHP and ETV1 expression was revealed between RA and untypical anaplastic anemia, iron deficiency anemia, and megaloblastic anemia patients. We propose that the technology of microarray may reveal the intrinsic molecular features and the expression levels of RNAHP, DDIT3, and ETV1 may provide useful markers for the diagnosis of MDS.
Adult↗
Xenopus Germes encodes a novel germ plasm-associated transcript.
A cDNA tag specific for the vegetal pole of early Xenopus embryo was used to isolate a novel cDNA using RACE technique. The corresponding mRNA demonstrated a localization pattern typical for the germ plasm-associated messages, and was, therefore, named Germes. The open reading frame of Germes encodes a predicted 68 kDa protein with two leucine zipper motifs and a putative EF-hand domain, but otherwise no substantial homology to known proteins. In situ hybridization analysis shows that Germes transcript localizes to the vegetal cortex via the mitochondrial cloud early in oogenesis and segregates with the germ plasm during early embryogenesis. Our data indicate that Germes is a novel germ plasm-specific RNA.
Amino Acid Sequence↗
Olfactory-specific cytochrome P-450. cDNA cloning of a novel neuroepithelial enzyme possibly involved in chemoreception.
We isolated cDNA clones for cytochrome P-450 genes expressed in the olfactory neuroepithelium by screening a corresponding rat cDNA library. Sequence analysis and RNA blot hybridization revealed a new cytochrome P-450, designated cytochrome P-450olf1, which is the first reported cytochrome P-450 mRNA uniquely expressed in the chemosensory organ. Cytochrome P-450olf1 shows intermediate level of sequence similarity (38-53% identity) to several liver cytochrome P-450 enzymes, suggesting that it belongs to the cytochrome P-450II family, but defines a new subfamily (cytochrome P-450IIG) within it. Cytochrome P-450II enzymes are known to process diverse organic compounds, including odorants. This, together with the specificity of cytochrome P-450olf1 to the sensory neuroepithelium, may indicate a role for this protein in olfactory reception.
Amino Acid Sequence↗
RNA profiling for biomarker discovery: practical considerations for limiting sample sizes.
We have compared microarray data generated on Affymetrix chips from standard (8 micrograms) or low (100 nanograms) amounts of total RNA. We evaluated the gene signals and gene fold-change estimates obtained from the two methods and validated a subset of the results by real time, polymerase chain reaction assays. The correlation of low RNA derived gene signals to gene signals obtained from standard RNA was poor for less to moderately abundant genes. Genes with high abundance showed better correlation in signals between the two methods. The signal correlation between the low RNA and standard RNA methods was improved by including a reference sample in the microarray analysis. In contrast, the fold-change estimates for genes were better correlated between the two methods regardless of the magnitude of gene signals. A reference sample based method is suggested for studies that would end up comparing gene signal data from a combination of low and standard RNA templates; no such referencing appears to be necessary when comparing fold-changes of gene expression between standard and low template reactions.
Biomarkers↗
Efficient RNA interference depends on global context of the target sequence: quantitative analysis of silencing efficiency using Eulerian graph representation of siRNA.
Several aspects of gene silencing by small interfering RNA duplexes (siRNA) influence the efficiency of the silencing. They can be divided into two categories, one covering the cell-specific factors and the other covering molecular factors of the RNA interference (RNAi). A prerequisite for sequence-based siRNA design is that hybridization thermodynamics is the dominant factor. Our assumption is that cell-specific parameters (cell line, degradation, cross-hybridization, target conformation, etc.) can be pooled into an average cellular factor. Our hypothesis is that the molecular basis of the positional dependence of siRNA-induced gene silencing is the uniqueness of context of a corresponding target sequence segment relative to all other such segments along the attacked RNA. We encode this context into descriptors derived from Eulerian graph representation of siRNAs and show that the descriptor based upon the contextual similarity and predicted thermodynamic stability correlates with the experimentally observed silencing efficiency of human lamin A/C gene. We further show that information encoded in this regression function is generalizable and can be used as a predictor of siRNA efficiency in unrelated genes (CD54 and PTEN). In summary, our method represents an evolution of siRNA design from the currently used algorithms which are only qualitative in nature.
Base Sequence↗
Directly labeled mRNA produces highly precise and unbiased differential gene expression data.
Microarray based gene expression studies allow simultaneous analysis of relative amounts of messenger RNA (mRNA) for thousands of genes using fluorescently labeled nucleic acid targets. Most common methods use enzymatic techniques, such as oligo-dT primed reverse transcription to produce labeled cDNA. These labeling methods have a number of shortcomings, including enzyme- introduced labeling and sequence bias, laborious protocols, high experiment-to-experiment variability and an inability to detect small changes in expression levels. Here, we describe a novel labeling methodology that uses platinum-linked cyanine dyes to directly chemically label mRNA from as little as 2 micro g of total RNA. We show that the gene expression data produced using the labeled mRNA method has very high precision, low error, no labeling bias and a dynamic range over several orders of magnitude. This allows a greater accuracy in the identification of differentially expressed genes and cuts down on the need for running too many replicate assays. Small changes in gene expression can now be detected in large-scale gene expression profiling assays using this simple, easy and quick procedure.
Carbocyanines↗
Novel transcript nort is a downstream target gene of the Notch signaling pathway in zebrafish.
The Notch signaling pathway plays important roles in the regulation of diverse developmental processes. Although many Notch-signal target genes with different specificities have been identified, their regulation and functions are not fully understood. Here, we conducted a microarray screen to search for novel downstream target genes of the Notch pathway in zebrafish. From the screen, we isolated nort (Notch-regulated transcript) as a transcript whose expression was reduced by the inhibition of Notch signaling. The expression level of nort increased when Notch signaling was activated. nort was expressed in hypoblast cells and the developing nervous system. We found its expression pattern to be similar to that of her4, but it showed some differences, at least in the anterior and posterior neural plate at the 3-somite stage. The nort transcript did not contain any long open-reading frame (ORF) of more than 300 nt, and its ORF-encoded sequence showed no significant homology with the proteins in databases. However, nort has one SPS (suppressor of hairless paired binding site) in its 5'-flanking region. These data suggest that nort is a putative noncoding RNA regulated by Notch signaling.
5' Flanking Region↗
Analysis of the in vitro secondary structure of cucumber mosaic virus satellite RNA.
The solution conformation of two variants of cucumber mosaic virus satellite RNA (CMV satRNA) was analyzed using several enzymatic and chemical probes. Ribonuclease T1 and nuclease S1 were used to map unpaired nucleotides, and nuclease V1 was used to detect double-stranded, or stacked, bases. Chemical probing with dimethylsulphate and diethylpyrocarbonate also identified unpaired and unstacked nucleotides, respectively. Modified or cleaved positions were identified by direct gel electrophoresis of radioactively labeled RNA, or by analysis of DNA sequence patterns generated by primer-extension with reverse transcriptase. Additional information was obtained by a gel-fractionation method under nondenaturing conditions for the identification of base paired fragments. On these data, a model for the in vitro secondary structure of CMV satRNA is proposed. Results support the existence of a complex structure with 51% of nucleotides involved in base pairs (40 G:C, 28 G:U, and 18 A:U pairs). Several structural elements, numbered I-VI, were defined, and interactions between separate domains are suggested. Comparisons of experimental data and a formerly reported secondary structure model for CMV satRNA support the validity of the structure we propose.
Base Composition↗
Characterization of a temperature-sensitive influenza B virus mutant defective in neuraminidase.
ts5, a temperature-sensitive mutant of influenza B virus, belongs to one of seven recombination groups. When the mutant infected MDCK cells at the nonpermissive temperature (37.5 degrees C), infectious virus was produced at very low levels compared with the yield at the permissive temperature (32 degrees C) and hemagglutinating and enzymatic activities were undetectable. However, viral protein synthesis and transport of hemagglutinin (HA) and neuraminidase (NA) to the cell surface were not affected. The NA was found as a monomer within cells even at 32 degrees C, in contrast to wild-type virus NA, existing mostly as an oligomer, but the mutant had oligomeric NA, like the wild-type virus. Its enzymatic activity was more thermolabile than that of wild-type virus. Despite the low yield, large aggregates of progeny virus particles were found to accumulate on the cell surface at the nonpermissive temperature, and these aggregates were broken by treatment with bacterial neuraminidase, with the concomitant appearance of hemagglutinating activity, suggesting that NA prevents the aggregation of progeny virus by removal of neuraminic acid from HA and cell receptor, allowing its release from the cells. Further treatment with trypsin resulted in the recovery of infectivity. When bacterial NA was added to the culture early in infection, many hemagglutinable infectious virus was produced. We also suggest that the removal of neuraminic acid from HA by NA is essential for the subsequent cleavage of HA by cellular protease. Nucleotide sequence analysis of RNA segment 6 revealed that ts5 encoded five amino acid changes in the NA molecule but not in NB.
Amino Acid Sequence↗
Identification of miRNA expression profile in middle ear cholesteatoma using small RNA-sequencing.
BACKGROUND: The present study aims to identify the differential miRNA expression profile in middle ear cholesteatoma and explore their potential roles in its pathogenesis. METHODS: Cholesteatoma and matched normal retroauricular skin tissue samples were collected from patients diagnosed with acquired middle ear cholesteatoma. The miRNA expression profiling was performed using small RNA sequencing, which further validated by quantitative real-time PCR (qRT-PCR). Target genes of differentially expressed miRNAs in cholesteatoma were predicted. The interaction network of 5 most significantly differentially expressed miRNAs was visualized using Cytoscape. Further Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analyses were processed to investigate the biological functions of miRNAs in cholesteatoma. RESULTS: The miRNA expression profile revealed 121 significantly differentially expressed miRNAs in cholesteatoma compared to normal skin tissues, with 56 upregulated and 65 downregulated. GO and KEGG pathway enrichment analyses suggested their significant roles in the pathogenesis of cholesteatoma. The interaction network of the the 2 most upregulated (hsa-miR-21-5p and hsa-miR-142-5p) and 3 most downregulated (hsa-miR-508-3p, hsa-miR-509-3p and hsa-miR-211-5p) miRNAs identified TGFBR2, MBNL1, and NFAT5 as potential key target genes in middle ear cholesteatoma. CONCLUSIONS: This study provides a comprehensive miRNA expression profile in middle ear cholesteatoma, which may aid in identifying therapeutic targets for its management.
Humans↗
Clinical and prognostic significance of detection of enteroviral RNA in the myocardium of patients with myocarditis or dilated cardiomyopathy.
BACKGROUND: Enteroviral RNA sequences have been demonstrated in the myocardium of patients with myocarditis or dilated cardiomyopathy from presentation to end-stage disease. The prognosis of heart muscle disease has not previously been evaluated in relation to the detection of enterovirus in myocardial biopsy tissue. METHODS AND RESULTS: We studied 123 consecutive patients with heart muscle disease prospectively. Multiple endomyocardial biopsy samples taken from all patients during diagnostic cardiac catheterization were classified histologically and were examined for enteroviral RNA by use of an enterovirus group-specific hybridization probe. Three enterovirus-negative patients with cardiac amyloidosis were excluded from subsequent analysis. Enteroviral RNA sequences were detectable in 41 (34%) of the remaining 120 patients (group A), while 79 (66%) had no virus detected (group B). The groups did not differ significantly in age, sex, symptomatic presentation, or hemodynamic characteristics; duration of symptoms was significantly shorter in group A (7.8 +/- 9.6 versus 14.9 +/- 19.0 months, P < .05). At follow-up (mean, 25 months; range, 11 to 50 months), patients from group A had an increased mortality compared with those in group B (25% versus 4%, respectively; P = .02). Mortality was also statistically greater in patients with symptomatic cardiac failure (P = .02), those with elevated left ventricular end-diastolic pressures (P = .03), and those in New York Heart Association functional classes III and IV (P = .05). Multivariate regression analysis, however, showed that only the presence of enterovirus RNA and symptomatic heart failure were of independent prognostic value. CONCLUSIONS: These data demonstrate that the detection of enterovirus RNA in the myocardium of patients with heart muscle disease at the time of initial investigation is associated with an adverse prognosis and that the presence of enterovirus RNA is an independent predictor of clinical outcome.
Adolescent↗
Possibilities and results in the wide-scale genomic analysis of inflammation. Looking for the child among many midwives.
Only recently, available microarray methods have been extended to the investigation of such complex pathophysiological conditions as inflammatory disease. Genome analysis gives the possibility of looking for susceptibility gene loci and their allelic combinations. By the analysis of mRNAs (transcriptome), novel players, functional groups of participants and regulatory pathways involved in the inflammation can be revealed. The possibility of comparing several samples is ideal to increase our knowledge about the kinetics of inflammation. The effects and post-receptor signalling events of individual pro- or anti-inflammatory mediators can also be tested. The use of human post mortem samples could yield otherwise inaccessible information on molecular mechanisms of chronic inflammatory diseases. However, the analysis and interpretation of a huge amount of data, the selection of appropriate experimental systems and time points may hide some pitfalls, which emphasize the need to confirm results with independent methods such as e.g. semi-quantitative Northern, real time PCR, RNA-ase protection assay as well as functional studies.
Acute-Phase Reaction↗
Comparison of the non-structural protein, NS1, of tick-borne and insect-borne orbiviruses.
The nucleotide sequence of RNA segment 6 of Broadhaven virus (BRDV), a tick-borne orbivirus, was determined principally from two overlapping cDNA clones and RNA end sequence analysis. The genome segment is 1714 base pairs in length and has a coding capacity for a protein of 537 amino acids, having a net charge of +4.0 at neutral pH. Comparison of the predicted amino acid sequence of BRDV RNA segment 6 with the NS1 sequence of insect-borne orbiviruses, bluetongue virus (BTV), African horse sickness virus (AHSV) and epizootic haemorrhagic disease virus (EHDV) of deer, revealed amino acid identities of 21, 22 and 21%, respectively. This compares with amino acid identities of 31 to 50% between the NS1 proteins of these gnat-transmitted orbiviruses. A recombinant baculovirus was produced containing a full-length clone of BRDV segment 6, which expressed a protein of 61 kD in infected Spodoptera frugiperda cells. Like other orbivirus NS1 proteins the expressed protein formed tubules similar to those produced in BRDV-infected BHK21 cells.
Amino Acid Sequence↗