High-density yeast-tiling array reveals previously undiscovered introns and extensive regulation of meiotic splicing

Abstract

Knowing gene structure is vital to understanding gene function, and accurate genome annotation is essential for understanding cellular function. To this end, we have developed a genome-wide assay for mapping introns in Saccharomyces cerevisiae. Using high-density tiling arrays, we compared wild-type yeast to a mutant deficient for intron degradation. Our method identified 76% of the known introns, confirmed 18 previously predicted introns, and revealed 9 formerly undiscovered introns. Furthermore, we discovered that all 13 meiosis-specific intronic yeast genes undergo regulated splicing, which provides posttranscriptional regulation of the genes involved in yeast cell differentiation. Moreover, we found that Ϸ16% of intronic genes in yeast are incompletely spliced during exponential growth in rich medium, which suggests that meiosis is not the only biological process regulated by splicing. Our tiling-array assay provides a snapshot of the spliced transcriptome in yeast. This robust methodology can be used to explore environmentally distinct splicing responses and should be readily adaptable to the study of other organisms, including humans. meiosis ͉ regulated splicing ͉ Saccharomyces cerevisiae I ntronic sequences provide numerous functional elements that direct pre-mRNA processing and alternative splicing. In the relatively simple eukaryote Saccharomyces cerevisiae, introns direct splicing (1), can increase gene expression (2), and, in specific cases, may contain small nucleolar RNAs (3). Additionally, introns in yeast can modulate translation posttranscriptionally through a process known as regulated splicing (4-7). During regulated splicing, yeast cells under certain conditions can limit intron splicing in specific genes, which, in turn, disrupts translation through frame-shifting and/or introduction of nonsense codons (4-7). Accurate mapping of introns is an essential first step to understanding RNA splicing and function.S. cerevisiae is an easily manipulatable eukaryote with a relatively small extensively studied genome that shares many core spliceosome functions with humans (1). Only 5% of S. cerevisiae genes are interrupted by introns (8, 9), and all introns are constitutively removed before translation (10). Because its genome is relatively small and well characterized, yeast serves as an ideal model organism for new technologies.Tiling DNA microarrays, comprised of overlapping, end-toend, or closely spaced DNA probes, have been used to map cellular transcription in a variety of organisms. Tiling-array data have improved gene annotation and revealed extensive transcription of noncoding RNAs (11-13). We used a high-density yeast-tiling array with overlapping probes, which provides a per-strand resolution of eight nucleotides, to research premRNA processing in yeast. The closely spaced probes allowed for accurate measurement of small transcriptional features, such as single exons and small introns.In this paper, we describe the results of a high-resolution microarray investigation of yeast splicing during...

Interactions between Mei4, Rec114, and other proteins required for meiotic DNA double-strand break formation in Saccharomyces cerevisiae

“…5c), and the expected sequence at the splice junction was confirmed by sequencing of the RT-PCR product (data not shown). Splicing of the REC102 message was also demonstrated by large-scale microarray and RT-PCR analyses in recent independent studies (Miura et al 2006;Juneau et al 2007). Interestingly, splicing was relatively inefficient in that the intron had been removed in only ∼70% of the amplified message from RNA samples isolated 6 hr after transfer to sporulation medium (Fig.…”

“…S1b, c). S. cerevisiae REC114 has an intron near the 3′ end Juneau et al 2007), a placement that is very rare in yeast (Lopez and Seraphin 1999;Lopez and Seraphin 2000). This intron splits the Cterminal conserved domain and is found in many of the REC114 homologs (of the homologs shown, only those from C. glabrata and S. castellii lack the intron) (arrow in Fig.…”

“…It has been suggested that this difference may simply reflect less opportunity (on an evolutionary time scale) or less selection pressure for meiotic transcripts to be subject to intron loss through reverse transcription and recombination ). An alternative possibility was suggested by the observation that the majority of meiotic introns (including the one in REC102) show meiosis-specific regulation of their splicing efficiency (Juneau et al 2007). In this model, there is selective pressure to maintain introns in meiotic genes because their inefficient splicing in vegetative cells serves as an additional layer of repression to prevent expression of proteins that might be deleterious in vegetative cells (Juneau et al 2007).…”

“…An alternative possibility was suggested by the observation that the majority of meiotic introns (including the one in REC102) show meiosis-specific regulation of their splicing efficiency (Juneau et al 2007). In this model, there is selective pressure to maintain introns in meiotic genes because their inefficient splicing in vegetative cells serves as an additional layer of repression to prevent expression of proteins that might be deleterious in vegetative cells (Juneau et al 2007). Notably in this regard, intron structure is conserved among the DSB proteins despite very poor overall amino acid conservation Henderson et al 2006).…”

Assessing Differential Expression Measurements by Highly Parallel Pyrosequencing and DNA Microarrays: A Comparative Study

“…All of them were 5¢ heterogeneity length variants. However, a comprehensive survey of the literature revealed that such unconventional splice variants had already been described elsewhere ( Juneau et al, 2007;Miura et al, 2006). Only new splice variants supported by, at least, a 10 · coverage were considered.…”