Refining the Message
Professors Jean Beggs and David Tollervey of the Wellcome Trust Centre for Cell Biology, University of Edinburgh, are using a combination of proteomics and genetics to study how RNA is made and how it functions in yeast cells. RNA molecules are crucial for the function of all types of cells. While genes store genetic information in the chemical sequence of DNA molecules, for this information to be used it must be copied into the related molecules called RNA…
What you need to know about RNA processing:
RNA is produced in the nucleus of the cell. The mRNA (messenger RNA) has to be processed and transported to the cytoplasm to produce proteins.
Introns and RNA Splicing
Some genes have their protein-coding information interrupted by non-coding sequences called introns. The intron will also be present in the RNA copy of the gene and must be removed by a process called RNA splicing before proteins can be produced.
Spliceosomes and RNA splicing
Spliceosomes are complexes that contain around 80 to 100 proteins and 5 small RNAs in addition to the pre-mRNA. The spliceosome is only one of many RNA-protein machines in the cell. In the splicing process, spliceosomes remove introns from messenger RNA precursors (pre-mRNAs). These are highly dynamic complexes and will undergo many rearrangements and changes in composition during the course of the splicing.
RibosomesRibosomes are the molecular machines that produce the protein products encoded by genes and messenger RNAs (mRNAs); they contain about 80 proteins and four ribosomal RNAs (rRNAs). The synthesis of ribosomes is one of the major metabolic pathways in all cells.
RNA molecules are crucial for the function of all types of cells. While genes store genetic information in the chemical sequence of DNA molecules, for this information to be used it must be copied into the related molecules called RNA. Many types of RNA thus act as genetic messengers, termed "mRNAs", which carry information from the genes that instruct the cell how to assemble the many different types of proteins needed for life. This process of making RNA is highly complex and involves many separate events that are carried out by a wide array of protein factors.
These processing events are important control steps in gene expression and can also increase the coding potential of the genome, as individual mRNAs can be processed in different ways, giving rise to the capacity to encode more proteins.
The common baker’s yeast (Saccharomyces cerevisiae) is widely used by biologists to analyse complex processes in cells because it is easy to grow and study in the laboratory. All of the DNA in yeast cells has been sequenced and therefore all the yeast genes are now known. As yeast and human cells are very similar at the single cell level, results obtained through studying yeast cells, which is usually easier than studying human cells, can help us to understand how human cells function. Therefore, yeast has been used to reveal the complex pathways of RNA production within cells and especially to reveal the interactions between different proteins involved in this process. Two particular techniques have been used by Beggs and Tollervey that provide valuable insights into how RNA is produced. The first involves clever in vivo screening methods to detect protein-protein and protein-nucleic acid interactions. Jean Beggs comments, "I have collaborated recently with Dr Pierre Legrain and colleagues (formerly working in the Institut Pasteur) to use the so-called "two-hybrid" screening strategy to identify interactions between yeast proteins involved in critical steps in the production of RNA. In this way we identified many novel proteins and uncovered unexpected links between different pathways affecting RNA molecules."
This screening methodology can now be carried out using robots to automate and thereby greatly speed up the laboratory procedures. In this way very large numbers of genes can be analysed, hence revealing vital clues about which proteins interact with which partners.
The second approach employed by Beggs and Tollervey involves using mass spectrometry to study yeast proteins in a similar way to that used in the study of human cell proteins (see articles by Lamond and Kolch). As Professor Tollervey explains, "We first use DNA technology to add a small chemical "tag" (known as a Tandem Affinity Purification (TAP) peptide) to the protein we want to study and then use this tag as a hook for purifying the protein along with all of the other proteins that bind to it. These interaction partner proteins are then identified using mass spectrometry."
These two approaches have produced large amounts of important new data that have helped improve our understanding of RNA production and other cell processes involving complex protein assemblies. These strategies have been exploited in a recent large international collaboration funded by the European Union and coordinated by Beggs and Tolllervey together with four other European research groups. For further information see; www.eurnomics.org.
This work points to the future directions of biological research. The more traditional, small-scale projects using the "bottom-up" approach - i.e. with individual research groups studying only one or two factors in isolation –are now being supplemented by "top-down" methods, which attempt to simultaneously analyse hundreds or even thousands of different proteins at once. These large-scale projects are often referred to as "Systems Biology", because their aim is to explain how the entire system of protein interactions within a cell operates.