Exploring the transcriptome of Aspergillus nidulans

For many organisms, ranging from pathogenic bacteria to humans, their DNA content, the genome, has been sequenced. This is a marvellous tool for us to improve our understanding of biology with far reaching consequences in fighting human disease, safeguarding and enhancing food production, developing new sources of energy and producing a wide array of products we need, ranging from medicines to fine chemicals. However, the usefulness of these genome sequences is limited by our understanding; it is not a trivial exercise to interpret the information they contain. For example it is often difficult to identify precisely where genes are located, where they begin and end, what features allow one gene to be highly expressed and another weakly. In order to help us interpret and fully exploit the genome sequences it is invaluable to be able to identify all the RNA molecules that the genome encodes, the transcriptome. This has recently become far more straightforward, with the advent of new sequencing technologies that allow us to obtain hundred of millions of short sequences very rapidly and relatively cheaply. These data can then be analysed by sophisticated computational techniques to build a detailed picture of the genomes function. From this we can establish all the different genes that are present, how they behave under different conditions and how they are organised. The aim of this project is to utilise these sequencing approaches to look at the transcriptome of a fungus, Aspergillus nidulans. Although this organism does occasionally cause disease, particularly in individuals suffering from a rare genetic disorder called Chronic Granulomatous disease, it is primarily employed as a model for understanding fundamental biological systems and in particular closely related species of major importance. These include: Aspergillus fumigatus which is an important allergen, often being associated with sick building syndrome, and the major cause of invasive aspergillosis, a life threatening infection in severely immune-compromised patients with a high mortality rate (25-90%). Unfortunately, the effectiveness of the currently available treatments is limited and diagnosis is problematic. Controlling Aspergillus flavus is important to prevent food spoilage, because of its ability to produce aflatoxin, the most potent carcinogen know. Aspergillus niger is the source of most citric acid used in soft drinks and is also used for both the production of proteins and as a source of enzymes for the food industry. Aspergillus oryzae is also used to manufacture of many traditional fermented Japanese food products, such as soy sauce and saki as well as in biotechnology. The aim of this project is therefore to develop a much greater appreciation of how the Aspergillus genome is organised and functions, allowing us to better understand the fundamental nature of these organisms. The potential is that by improving our understanding of Aspergillus biology we will be better equipped to detect, control and eradicate them where necessary and exploit their potential in developing new biotechnological processes and products.

We propose to use next generation sequencing to characterise the transcriptome of Aspergillus nidulans. This will be conducted after growth under various conditions and for specific cell types. Additionally we will investigate the effect of various mutations on the RNA profile, including the disruption of RNAi, nonsense mediated decay, nuclear exosome function and the COMPASS complex, which catalyzes the methylation of Lys4 of histone 3. Four distinct sequencing approaches will be employed. Firstly, whole transcriptome sequencing will be utilised to characterise the mRNA. Bioinformatic analysis of these data will be used to identify splice sites (including differential splicing) and 3' ends of polyadenylated genes. Modified 5' RATE will be employed to define the transcription start sites. Small RNAs will also be isolated and sequenced. Finally we will sequence RNA associated with polysomes, to discern the level of differential regulation and if it correlates with specific groups of genes based on function (GO terms), or the inclusion of particular features such as short upstream ORFs. Detailed analyses will be conducted to validate the observations made from the sequencing approach. Downstream analysis will focus on testing various aspects. The function of up to ten antisense and other non-conding RNAs will be investigated. Specific features identified by computational analysis as being associated with highly expressed genes or defining gene structure (eg 3' end formation) will be tested functionally utilising reporter constructs. The aim will be to develop rational design strategies for synthetic genes and expression systems. We will deposit the sequence data in public databases and transfer it to CADRE and AspGD for incorporation into annotation pipelines. Expression data will also be made viewable through the Ensembl genome browser.

The outcome of this research will benefit scientific knowledge and also, indirectly, applied research. Our development of improved technology and bioinformatic methods will assist other researchers. Staff working on this project will develop skills in cutting edge technologies that are in demand by industry. This will include training of both post-doctoral researchers and at least one post-graduate student in next generation sequencing technology and bioinformatic analysis. One major impact will be significantly improved annotation of other filamentous fungal genomes, including economically and medically important species. Dissemination of this information will be facilitated via the public databases: AspGD, CADRE and Ensembl. In the long term the significant advances from this study will facilitate will help in developing: - New biotechnological process utilising fungi as cell factories - New leads to the identification of novel genes/proteins that cane be exploited - A greater understanding of how fungi adapt to environmental change, which is fundamental to our understanding of pathogenicity. This work will therefore influence research in this area, potentially leading in the long term to novel therapies or diagnostic techniques. The work in this project is thus important to underpin the BBSRC's strategic aim to underpin practical solutions to major challenges that include food security and the control of infectious diseases, which is particularly relevant to individuals with long term illness and immune suppression, including transplant patients and the elderly. Therefore because of its nature, aspects of this project make it relevant to a wide range of the BBSRC's strategic priorities including: - Ageing research: lifelong health and wellbeing - Bioenergy - Global security - Synthetic biology - Animal health. It underlies responses of organisms to environmental and developmental change, and is of strategic importance in living with environmental change which is one of the BBSRC's research priorities. It is also fully in line with BBSRC's professed aims to exploit and extending genomic resources, utilising high throughput sequencing and bioinformatic analysis.