Genomic-scale quantitative analysis of yeast pre-mRNA splicing: implications for splice-site recognition.

The availability of the complete sequence of the genome of Saccharomyces cerevisiae (Goffeau et al+, 1996) and other organisms has provided a substantial amount of information on their chromosomal organization+ We need now to understand the coordinated expression of the many genes present in a given genome and the function of the various encoded proteins+ Microarrays (DNA chips) offer the opportunity to analyze nucleic acids at the genomic scale and have been used to perform global gene expression studies (Lander, 1999)+ However, these techniques also have the potential to give us a new perspective on posttranscriptional processes involved in the regulation of gene expression+We provide here a striking example through the investigation of yeast pre-mRNA splicing+Our analysis demonstrates that pre-mRNA splicing is quantitatively a much more important process in this species than previously thought and shows that splicing-signal conservation is correlated with transcription efficiency+ It has often been suggested that pre-mRNA splicing is a minor process in yeast because only a tiny proportion of yeast genes contains introns (Dujon, 1996)+ However, the real question is what fraction of all transcripts is spliced? In other words, what proportion of total transcripts is dealt with by the splicing machinery? To answer this question quantitatively, we first created a database of known and predicted yeast introns+ For this purpose, we retrieved relevant information from several databases (SGD: Saccharomyces Genome Database,YPD:Yeast Protein Database,MIPS:Munich Information center for Protein Sequences, EMBL: European Molecular Biology Laboratory) and recent articles (Long et al+, 1997; Burge et al+, 1999; Spingola et al+, 1999)+ This information was manually edited to remove entries with annotation errors or where experimental evidence indicated the absence of an intron+ We ended up with a list of 253 introns (available at http://www+embl-heidelberg+de/ExternalInfo/seraphin/ yidb+html) present in 248 genes+ The presence of intron is not uniform among the different class of genes; in particular, ;70% of genes encoding for ribosomal protein contain an intervening sequence (Woolford, 1989; Rymond & Rosbash, 1992; Planta & Mager, 1998; Spingola et al+, 1999)+ For 228 of these genes, a transcription frequency (i+e+, number of transcripts produced per unit of time) has been determined (Holstege et al+, 1998)+ We could therefore estimate the fraction of pre-mRNAs among all transcribed cellular RNAs+ This revealed that although only 3+8% of the genes contain introns, 27+1% of the transcripts synthesized by the cell are spliced+ This large difference occurs because a large fraction of intron-containing RNAs, notably ribosomal proteins premRNAs, are highly expressed and demonstrates that pre-mRNA splicing is much more preponderant in yeast than previously suspected+ Our intron database also allowed us to examine the conservation of pre-mRNA splicing signals present at the 59 splice site, branchpoint, and 39 splice site by calculating the frequencies of each nucleotide in these regions+ To assess the significance of this analysis, we performed x2 tests, taking into account the biased composition of yeast coding sequences (see Fig+ 1)+ This confirmed the importance of previously known splicing signals and demonstrated significant conservation of the flanking sequences (Fig+ 1)+ Interestingly, tabulating the representation of the corresponding nucleotides in the pool of cellular pre-mRNAs, taking expression levels into account, reveals greater representation of the less-conserved positions (Fig+ 1)+ This indicates that the selection pressure on splicing signals is stronger for highly expressed transcripts+ Notably, our analysis revealed conservation of As at positions 22 to 24 of Reprint requests to: Bertrand Seraphin, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117, Heidelberg, Germany; e-mail: Seraphin@EMBL-Heidelberg+de+ RNA (1999), 5:1135–1137+ Cambridge University Press+ Printed in the USA+ Copyright © 1999 RNA Society+