MicroRNAs (miRNAs) are small noncoding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents that were enriched in neuronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the differences in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units, and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.
Neurospora crassa is a central organism in the history of twentieth-century genetics, biochemistry and molecular biology. Here, we report a high-quality draft sequence of the N. crassa genome. The approximately 40-megabase genome encodes about 10,000 protein-coding genes-more than twice as many as in the fission yeast Schizosaccharomyces pombe and only about 25% fewer than in the fruitfly Drosophila melanogaster. Analysis of the gene set yields insights into unexpected aspects of Neurospora biology including the identification of genes potentially associated with red light photobiology, genes implicated in secondary metabolism, and important differences in Ca(2+) signalling as compared with plants and animals. Neurospora possesses the widest array of genome defence mechanisms known for any eukaryotic organism, including a process unique to fungi called repeat-induced point mutation (RIP). Genome analysis suggests that RIP has had a profound impact on genome evolution, greatly slowing the creation of new genes through genomic duplication and resulting in a genome with an unusually low proportion of closely related genes
Although it is known that the human genome contains hundreds of microRNA (miRNA) genes and that each miRNA can regulate a large number of mRNA targets, the overall effect of miRNAs on mRNA tissue profiles has not been systematically elucidated. Here, we show that predicted human mRNA targets of several highly tissue-specific miRNAs are typically expressed in the same tissue as the miRNA but at significantly lower levels than in tissues where the miRNA is not present. Conversely, highly expressed genes are often enriched in mRNAs that do not have the recognition motifs for the miRNAs expressed in these tissues. Together, our data support the hypothesis that miRNA expression broadly contributes to tissue specificity of mRNA expression in many human tissues. Based on these insights, we apply a computational tool to directly correlate 3 UTR motifs with changes in mRNA levels upon miRNA overexpression or knockdown. We show that this tool can identify functionally important 3 UTR motifs without cross-species comparison.gene expression ͉ microarray ͉ posttranscriptional control
Up to 36% of Neurospora crassa transformants showing an albino phenotype were recovered by transforming a wild-type strain with different portions of the carotenogenic albino-3 (al-3) and albino-1 (al-1) genes. The presence of the exogenous sequences (which were randomly integrated in ectopic locations) provoked a severe impairment in the expression of the endogenous al-1 or al-3 genes. This phenomenon, which we have termed 'quelling', was found to be spontaneously and progressively reversible, leading to wild-type or intermediate phenotypes. The phenotypic reversion is characterized by a progressive release of the transcriptional inhibition and seems to correlate with a reduction of the number of the ectopic integrated sequences. Moreover, quelling appears to be monodirectional, as, once relieved, it cannot take place again, despite the continuing presence of some of the ectopic sequences in the genome.
The Neurospora crassa blind mutant white collar‐1 (wc‐1) is pleiotropically defective in all blue light‐induced phenomena, establishing a role for the wc‐1 gene product in the signal transduction pathway. We report the cloning of the wc‐1 gene isolated by chromosome walking and mutant complementation. The elucidation of the wc‐1 gene product provides a key piece of the blue light signal transduction puzzle. The wc‐1 gene encodes a 125 kDa protein whose encoded motifs include a single class four, zinc finger DNA binding domain and a glutamine‐rich putative transcription activation domain. We demonstrate that the wc‐1 zinc finger domain, expressed in Escherichia coli, is able to bind specifically to the promoter of a blue light‐regulated gene of Neurospora using an in vitro gel retardation assay. Furthermore, we show that wc‐1 gene expression is autoregulated and is transcriptionally induced by blue light irradiation.
In plants and fungi, the introduction of transgenes can lead to post-transcriptional gene silencing. This phenomenon, in which expression of the transgene and of endogenous genes containing sequences homologous to the transgene can be blocked, is involved in virus resistance and genome maintenance. Transgene-induced gene silencing has been termed quelling in Neurospora crassa and co-suppression in plants. Quelling-defective (qde) mutants of N. crassa, in which transgene-induced gene silencing is impaired, have been isolated. Here we report the cloning of qde-1, the first cellular component of the gene-silencing mechanism to be isolated, which defines a new gene family conserved among different species including plants, animals and fungi. The qde-1 gene product is similar to an RNA-dependent RNA polymerase found in the tomato. The identification of qde-1 strongly supports models that implicate an RNA-dependent RNA polymerase in the post-transcriptional gene-silencing mechanism. The presence of qde-1 homologues in a variety of species of plants and fungi indicates that a conserved gene-silencing mechanism may exist, which could have evolved to preserve genome integrity and to protect the genome against naturally occurring transposons and viruses.
A saturating genetic dissection of ‘blind’ mutants in Neurospora crassa has identified a total of two non‐redundant loci (wc‐1 and wc‐2) each of which is required for blue‐light perception/signal transduction. Previously, we demonstrated that WC1 is a putative zinc finger transcription factor able to bind specifically to a light‐regulated promoter. Here, we present the cloning and characterization of the wc‐2 gene. We demonstrate using mutation analysis and in vitro DNA‐binding assays that WC2, the second partner of this light signal transduction system, encodes a functional zinc finger DNA‐binding protein with putative PAS dimerization and transcription activation domains. This molecular genetic dissection of the second of two components of this light signal transduction system has enabled us to devise a model whereby WC1 and WC2 are proposed to interact via homologous PAS domains, bind to promoters of light‐regulated genes and activate transcription. As such, this study provides the first insight into two co‐operating partners in blue‐light signal transduction in any organism and describes the molecular tools with which to dissect this enigmatic process.
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