A comparative genome-wide study of ncRNAs in trypanosomatids

<p>Abstract</p> <p>Background</p> <p>Recent studies have provided extensive evidence for multitudes of non-coding RNA (ncRNA) transcripts in a wide range of eukaryotic genomes. ncRNAs are emerging as key players in multiple layers of cellular regulation. With the availability of many whole genome sequences, comparative analysis has become a powerful tool to identify ncRNA molecules. In this study, we performed a systematic genome-wide in silico screen to search for novel small ncRNAs in the genome of <it>Trypanosoma brucei </it>using techniques of comparative genomics.</p> <p>Results</p> <p>In this study, we identified by comparative genomics, and validated by experimental analysis several novel ncRNAs that are conserved across multiple trypanosomatid genomes. When tested on known ncRNAs, our procedure was capable of finding almost half of the known repertoire through homology over six genomes, and about two-thirds of the known sequences were found in at least four genomes. After filtering, 72 conserved unannotated sequences in at least four genomes were found, 29 of which, ranging in size from 30 to 392 nts, were conserved in all six genomes. Fifty of the 72 candidates in the final set were chosen for experimental validation. Eighteen of the 50 (36%) were shown to be expressed, and for 11 of them a distinct expression product was detected, suggesting that they are short ncRNAs. Using functional experimental assays, five of the candidates were shown to be novel H/ACA and C/D snoRNAs; these included three sequences that appear as singletons in the genome, unlike previously identified snoRNA molecules that are found in clusters. The other candidates appear to be novel ncRNA molecules, and their function is, as yet, unknown.</p> <p>Conclusions</p> <p>Using comparative genomic techniques, we predicted 72 sequences as ncRNA candidates in <it>T. brucei</it>. The expression of 50 candidates was tested in laboratory experiments. This resulted in the discovery of 11 novel short ncRNAs in procyclic stage <it>T. brucei</it>, which have homologues in the other trypansomatids. A few of these molecules are snoRNAs, but most of them are novel ncRNA molecules. Based on this study, our analysis suggests that the total number of ncRNAs in trypanosomatids is in the range of several hundred.</p

Non-Coding RNA Prediction and Verification in Saccharomyces cerevisiae

Non-coding RNA (ncRNA) play an important and varied role in cellular function. A significant amount of research has been devoted to computational prediction of these genes from genomic sequence, but the ability to do so has remained elusive due to a lack of apparent genomic features. In this work, thermodynamic stability of ncRNA structural elements, as summarized in a Z-score, is used to predict ncRNA in the yeast Saccharomyces cerevisiae. This analysis was coupled with comparative genomics to search for ncRNA genes on chromosome six of S. cerevisiae and S. bayanus. Sets of positive and negative control genes were evaluated to determine the efficacy of thermodynamic stability for discriminating ncRNA from background sequence. The effect of window sizes and step sizes on the sensitivity of ncRNA identification was also explored. Non-coding RNA gene candidates, common to both S. cerevisiae and S. bayanus, were verified using northern blot analysis, rapid amplification of cDNA ends (RACE), and publicly available cDNA library data. Four ncRNA transcripts are well supported by experimental data (RUF10, RUF11, RUF12, RUF13), while one additional putative ncRNA transcript is well supported but the data are not entirely conclusive. Six candidates appear to be structural elements in 5′ or 3′ untranslated regions of annotated protein-coding genes. This work shows that thermodynamic stability, coupled with comparative genomics, can be used to predict ncRNA with significant structural elements

A Cytosine Methyltransferase Homologue Is Essential for Sexual Development in Aspergillus nidulans

Background: The genome defense processes RIP (repeat-induced point mutation) in the filamentous fungus Neurospora crassa, and MIP (methylation induced premeiotically) in the fungus Ascobolus immersus depend on proteins with DNA methyltransferase (DMT) domains. Nevertheless, these proteins, RID and Masc1, respectively, have not been demonstrated to have DMT activity. We discovered a close homologue in Aspergillus nidulans, a fungus thought to have no methylation and no genome defense system comparable to RIP or MIP. Principal Findings: We report the cloning and characterization of the DNA methyltransferase homologue A (dmtA) gene from Aspergillus nidulans. We found that the dmtA locus encodes both a sense (dmtA) and an anti-sense transcript (tmdA). Both transcripts are expressed in vegetative, conidial and sexual tissues. We determined that dmtA, but not tmdA, is required for early sexual development and formation of viable ascospores. We also tested if DNA methylation accumulated in any of the dmtA/tmdA mutants we constructed, and found that in both asexual and sexual tissues, these mutants, just like wild-type strains, appear devoid of DNA methylation. Conclusions/Significance: Our results demonstrate that a DMT homologue closely related to proteins implicated in RIP and MIP has an essential developmental function in a fungus that appears to lack both DNA methylation and RIP or MIP. It remains formally possible that DmtA is a bona fide DMT, responsible for trace, undetected DNA methylation that i

Regional patterns of gene expression in human and chimpanzee brains

We have analyzed gene expression in various brain regions of humans and chimpanzees. Within both human and chimpanzee individuals, the transcriptomes of the cerebral cortex are very similar to each other and differ more between individuals than among regions within an individual. In contrast, the transcriptomes of the cerebral cortex, the caudate nucleus, and the cerebellum differ substantially from each other. Between humans and chimpanzees, 10% of genes differ in their expression in at least one region of the brain. The majority of these expression differences are shared among all brain regions. Whereas genes encoding proteins involved in signal transduction and cell differentiation differ significantly between brain regions within individuals, no such pattern is seen between the species. However, a subset of genes that show expression differences between humans and chimpanzees are distributed nonrandomly across the genome. Furthermore, genes that show an elevated expression level in humans are statistically significantly enriched in regions that are recently duplicated in humans

Fungal RNA Biology

This book presents an overview of the RNA networks controlling gene expression in fungi highlighting the remaining questions and future challenges in this area. It covers several aspects of the RNA-mediated mechanisms that regulate gene expression in model yeasts and filamentous fungi, organisms of great importance for industry, medicine and agriculture. It is estimated that there are more than one million fungal species on the Earth. Despite their diversity (saprophytic, parasitic and mutualistic), fungi share common features distinctive from plants and animals and have been grouped taxonomically as an independent eukaryotic kingdom. In this book, 15 chapters written by experts in their fields cover the RNA-dependent processes that take place in a fungal cell ranging from formation of coding and non-coding RNAs to mRNA translation, ribosomal RNA biogenesis, gene silencing, RNA editing and epigenetic regulation