Regulated transcript stability

Now that the genetic blueprint of many organisms has been deciphered, the next big challenge in Biology is to understand how the instructions are used to make a living creature. This highlights the subject of gene regulation, the study of which genes are in use at any time. One of the least understood parts is how the cell quickly gets rid of working copies of genes (the messenger RNA, mRNA, transcripts) once they are no longer needed. Several pathways are known that degrade mRNA, but the controls to protect mRNA that is still required while swiftly removing unwanted mRNA are little understood. It is likely that this fundamental level of gene control will have similar features in all organisms, with signalling and control systems exquisitely adapted for group of genes or even individual important genes according to the processes in which they are involved. Controlled mRNA degradation is a major regulatory mechanism in nitrogen metabolism in the filamentous fungus Aspergillus nidulans. We have recently identified the molecule (glutamine) that signals accelerated decay of some, but not all, mRNAs involved in nitrogen metabolism when there is sufficient good-quality nitrogen source available to the fungus. An even more exciting aspect is the discovery that a second molecule (nitrate) opposes this signal for specific mRNAs. It therefore provides an excellent way to identify and characterise the components that regulate mRNA stability. This well-studied system has all the genetic, information and technical resources to tackle the problem of how one mRNA remains stable while another is destroyed within minutes as the fungus responds to different nitrogen sources. The essential first step in mRNA degradation is to remove the poly(A) tail from the molecule. Enzyme complexes, highly conserved among all forms of life, contain proteins that carry out this function although the details remain to be uncovered. Based on our initial experiments, we propose that one protein, Ccr4p, is the main deadenylase for general mRNA decay, while another, Pop2p, is needed in specific regulated pathways. We will confirm and further characterise these proteins within this project, including how their activity against specific mRNAs is inhibited by the specific signal nitrate. Additional proteins must be required to regulate degradation and we have identified three promising candidates from initial experiments and bioinformatics analysis. We will characterise their functions and also carry out experiments to identify further regulatory proteins. Monitoring the half-lives of individual mRNAs is one of our basic techniques. DNA microarrays will let us see the global effects of these regulatory proteins. However, we will also use methodologies from classical genetic analysis, site-specific mutagenesis, protein tagging, two and three hybrid analysis and co-immunoprecipitation to achieve our goals. One further approach that we will use is real-time imaging to gain new insights into the spatial aspects of mRNA turnover, in collaboration with laboratories that are very experienced with cell imaging and the use of fluorescently tagged proteins in A. nidulans. Research into the co-ordination of mRNA degradation as part of gene regulation and signalling is in its infancy and this elegant system within an amenable organism will allow us to make a significant contribution.

Transcript stability is a major determinant of gene expression across eukaryotes and it is becoming increasingly apparent that degradation rates for many transcripts are precisely regulated. Significant advances have been made in determining key cellular components in these degradation processes and several RNA binding proteins have been characterised which participate in regulation. However, there are few good model systems where specific signals are known to affect transcript stability and generally such systems have been described for specific genes rather than gene networks. Recently, we have shown that in Aspergillus nidulans a significant subset of genes involved in nitrogen metabolism is regulated at the level of transcript stability / such that the transcripts are rapidly degraded in the presence of high intracellular Gln concentrations. This regulatory process is superimposed on a well characterised mechanism that acts at the level of transcript initiation. For at least two genes involved in nitrogen metabolism, a more sophisticated regulation is in place, where the specific substrate (nitrate) is able to counteract the Gln signal and stabilise transcription. Our preliminary work has established that two specific deadenylases are involved and for the first time we have clearly demonstrated that these two well conserved proteins have distinct roles, the Ccr4p orthologue being responsible for basal degradation and the Pop2p orthologue directing signalled degradation. Additionally we have identified a number of RNA binding proteins that are involved in promoting specific deadenylase activities, both of which must be inhibited for nitrate stabilisation. We propose to exploit this very tractable system utilising molecular genetics, transcriptomics, proteomics and cell biology to address key questions relating to how degradation and in particular deadenylation is regulated in eukaryotes.