Enabling High Throughput 5'end Determination for Eukaryotic Transcriptomes

The information contained in the genes of living cells has to be converted into cellular components that form structures and enable biochemical reactions to take place. This process is called gene expression and it is vital to all life. Gene expression comprises two main steps, called transcription and translation. In transcription, the information in the DNA sequences of the genes is converted into equivalent sequences in so-called messenger RNA (mRNA) molecules. In translation, the mRNA molecules are 'read' by a large molecular structure called the ribosome, which uses the information to dictate the synthesis of proteins. This short project will help us understand how the use of the information encoded in the mRNA molecules is controlled. In order to be able to do this, we need to have detailed information about the early part of each mRNA. Sequencing projects have not provided this information. This project will develop a new procedure that will be capable of obtaining this information for every mRNA in a chosen organism.

The number of completed genome sequences is increasing at a rapid rate, and the transcriptomes of these organisms are being studied in a wide range of expression profiling experiments. Genomic studies of this kind have had a great impact on biological research, enabling so-called 'global' approaches to the study of gene structure and function. However, a considerable amount of information is still missing from these genome databases, since they generally do not include characterisation of the untranslated regions of the cellular mRNAs. Indeed, the 5' and 3' ends of the great majority of mRNAs remain experimentally undefined. Since the UTRs play important roles in the posttranscriptional control of gene expression, this represents a significant deficiency in the genomic information currently available to the community. This problem is particularly significant for eukaryotes, since almost all of their mRNAs are generally monocistronic with the structure: 5'UTR / single main ORF / 3'UTR. A further deficiency in current genome databases is the lack of definitive characterisation of the number of protein-encoding ORFs. In the current proposal we describe a novel procedure that will, in the first instance, be applied to the transcriptome of Saccharomyces cerevisiae. It will also have wider application as the basis for similar studies on other organisms. This proof-of-principle project will establish the starting point for applying a semi-automated, high-throughput version of the procedure to whole transcriptomes in the future.