| Research Focus Splicing goes global J. David Barrass and Jean D. Beggs Wellcome Trust Centre for Cell Biology, University of Edinburgh, King’s Buildings, Mayfield Road, Edinburgh EH9 3JR, UK Transcriptomics, the analysis of the complement of mRNAs transcribed from a cell’s genome, currently focuses mainly on mature, processed mRNAs. However, posttranscriptional processing of primary transcripts can significantly affect both the quantity and the struc- ture of the mature mRNAs and therefore of the protein products. Recently, the development of an intron- specific microarray has permitted a preliminary analysis of the splicing of all intron-containing transcripts in Sac- charomyces cerevisiae. Here, we discuss the findings and what might be learned from this kind of approach. Microarrays have been very useful in the study of TRAN- SCRIPTOMES (see Glossary). However, as generally used, they do not assay the processing of transcripts. For example, microarrays that contain long cDNAs or PCR products as PROBES do not necessarily distinguish differ- ently spliced mRNAs because they detect all mRNAs that have sufficient complementarity, even if they are not identical. Microarrays that use short oligonucleotide probes, with only one probe per gene, normally detect only one EXON in a eukaryotic transcript, and differently processed products of the same gene will not be distin- guished unless the probe happens to span a SPLICE JUNCTION. Even Affymetrix microarrays, which contain multiple oligo probes per gene, are not designed speci- fically to distinguish among spliced, unspliced and ALTERNATIVELY SPLICED transcripts, although some infor- mation about tissue-specific splice variants can be gleaned from the relative abundance of different regions of a transcript [1]. Recently, Clark et al. [2] have developed a microarray to examine the processing of pre-mRNAs in the budding yeast Saccharomyces cerevisiae. This simple eukaryote is a good model organism for such studies, because its nuclear genome has only , 250 INTRON-containing genes, a number easily within the capacity of a microarray analysis. Although only 3.8% of S. cerevisiae genes contain introns, many of these are highly expressed and account for a disproportionate 27% of the mRNAs [3,4]. Therefore, intron removal is an important activity in S. cerevisiae. The components of the SPLICEOSOME have been exten- sively studied in this organism [5], and many mutations that affect pre-mRNA splicing factors have been identified, offering huge genetic potential for analysing spliceosome function. Because both the mechanism of splicing and the splicing machinery are highly conserved throughout eukaryotes, knowledge of yeast splicing could even provide insights into splicing defects that are implicated in human disease. In the new splicing microarray there are three kinds of oligonucleotide probe per gene (Fig. 1). One probe detects the presence of an intron, the second covers the splice junction to monitor correct formation of the mature mRNA, and, as a control, the third probe detects the 3 0 exon, which is present in both the spliced and the unspliced RNAs. In analyses of splicing efficiency, changes in the levels of transcripts due to transcription or degradation must be corrected for and this is done by dividing the intron and splice junction signals by the corresponding 3 0 exon signal (as a measure of the total amount of that transcript). These normalized values are referred to as the intron accumulation (IA) index and the splice junction (SJ) index, respectively. The IA index is a measure of the accumulation of intron-containing species, whereas the SJ index reflects the relative level of correctly spliced mRNA. There can be two general, but related, aims of this type of intron-specific microarray analysis. First, to determine which factors or circumstances affect pre-mRNA splicing, and second, to identify the various processed RNA species produced. The former is the easier objective and the aim of the work described by Clark et al. [2]. They examined the effects of mutations in genes believed to be involved in pre- mRNA splicing in S. cerevisiae. If a mutation affects splicing, there will be an accumulation of unspliced or Glossary Alternative splicing: The coding capacity of a gene can be increased by including or excluding from the mature transcript some portion(s) of the gene that could form the final protein. This is done at the stage of RNA splicing where introns can be included in the RNA and/or exons (or parts of exons) excluded. The range of proteins that an organism can produce can be greatly expanded in this way. Exon: That part of a gene that is represented in a mature, spliced mRNA. An exon in one mRNA can be excluded from another mRNA transcribed from the same gene (see alternative splicing). Intron: That part of a gene that is removed from the primary transcript during the RNA splicing process, so it is not present in the mature mRNA. It does not generally encode protein sequence (see alternative splicing). Proteome: The protein complement expressed by a genome. Unlike the genome, this changes in response to external and internal events, such as available nutrients or the development of a cell. Probe: The nucleic acid species that confers specificity in a hybridization reaction. For a conventional molecular biology hybridization, such as a Southern blot, this is a labelled nucleic acid, generally DNA in solution. For a microarray, this is an unlabelled cDNA or DNA oligonucleotide firmly attached to solid support. Splice junction: A region in the mRNA where two exons are joined. Spliceosome: The machinery for processing the pre-mRNA. Target: The species that is detected by a probe. For a Southern blot this is attached to the membrane; for a microarray this is in solution. Transcriptome: The complement of mRNAs transcribed from a cell’s genome. Corresponding author: Jean D. Beggs (jbeggs@ed.ac.uk). Update TRENDS in Genetics Vol.19 No.6 June 2003 295 http://tigs.trends.com