In vitro evolution facilitated the identification of a highly stable, structurally homogeneous mutant RNA that was readily crystallizable. Analysis of the structure suggests that improving RNA secondary structure can stabilize tertiary structure and perhaps promote crystallization. In addition, the higher resolution model provides new details of metal ion-RNA interactions and identifies a core of ordered water molecules that may be integral to RNA tertiary structure formation.

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...

The purpose of introns in the architecturally simple genome of Saccharomyces cerevisiae is not well understood. To assay the functional relevance of introns, a series of computational analyses and several detailed deletion studies were completed on the intronic genes of S. cerevisiae. Mining existing data from genomewide studies on yeast revealed that intron-containing genes produce more RNA and more protein and are more likely to be haplo-insufficient than nonintronic genes. These observations for all intronic genes held true for distinct subsets of genes including ribosomal, nonribosomal, duplicated, and nonduplicated. Corroborating the result of computational analyses, deletion of introns from three essential genes decreased cellular RNA levels and caused measurable growth defects. These data provide evidence that introns improve transcriptional and translational yield and are required for competitive growth of yeast. T HE genes of complex organisms depend on intronsto provide regulatory sequences that allow for accurate pre-mRNA processing and alternative splicing. In multicellular organisms most genes contain at least one intron, usually more. In humans, for instance, 94% of the genes are interrupted by, on average, seven introns (Lander et al. 2001;Venter et al. 2001). Although splicing is closely coupled to several other processes during gene expression, it is still widely thought that the primary fitness benefits that introns confer to a species are through improved evolution via exon shuffling and increased proteome complexity by alternative splicing. On the basis of our observations we propose that introns confer an additional advantage: they improve the transcriptional and translational output of the genes they populate.The spliceosome, which removes introns from the coding mRNA, is a large cellular complex containing hundreds of proteins and at least five small nuclear RNAs. It is closely coupled to, and in some cases directly interacts with, the proteins responsible for transcription, capping, polyadenylation, RNA export, and nonsensemediated decay (Maniatis and Reed 2002). Given the extensive coupling of splicing with mRNA metabolism, it is not surprising that removing the introns from genes in higher eukaryotes (where intron-containing genes predominate) disrupts mRNA synthesis and often lowers cytoplasmic mRNA levels. The question arises: Are the introns directly responsible for increasing gene expression or does their removal act indirectly, by simply derailing the mRNA synthesis assembly line? Some examples in metazoans support a direct role in expression: introns containing transcriptional enhancers have been identified (Sleckman et al. 1996) and one group showed that removing introns from a gene disrupts nucleosome binding (Liu et al. 1995). There is, however, no consensus that introns serve to increase gene expression. To investigate the role that introns may play in cellular fitness we studied their genetic contribution to the fitness of Saccharomyces cerevisiae.In contrast to multi...

It is well established that higher eukaryotes use alternative splicing to increase proteome complexity. In contrast, Saccharomyces cerevisiae, a single-cell eukaryote, conducts predominantly regulated splicing through retention of nonfunctional introns. In this article we describe our discovery of a functional intron in the PTC7 (YHR076W) gene that can be alternatively spliced to create two mRNAs that code for distinct proteins. These two proteins localize to different cellular compartments and have distinct cellular roles. The protein translated from the spliced mRNA localizes to the mitochondria and its expression is carbon-source dependent. In comparison, the protein translated from the unspliced mRNA contains a transmembrane domain, localizes to the nuclear envelope, and mediates the toxic effects of Latrunculin A exposure. In conclusion, we identified a definitive example of functional alternative splicing in S. cerevisiae that confers a measurable fitness benefit. I N higher eukaryotes alternative splicing is pervasive; in humans the majority of genes are alternatively spliced to form multiple proteins (Modrek et al. 2001;Johnson et al. 2003). In contrast, only 5% of the genes in Saccharomyces cerevisiae contain introns and .95% of those intron-containing genes possess only a single intron (Nash et al. 2007). The simple architecture of the yeast genome constrains its ability to utilize alternative splicing and has encouraged the view that alternative splicing is absent in S. cerevisiae. Currently no conclusive examples of functional alternative splicing exist; most confirmed instances of alternative splicing in yeast downregulate gene expression, a process that is often referred to as ''regulated splicing.'' In this process, nonfunctional introns are not spliced out of the transcript and premature stop codons are included in the fully processed mRNA. The stop codons activate the nonsense-mediated decay (NMD) pathway and the mRNA is degraded before it can be translated (Gonzalez et al. 2001).The transition from vegetative growth to meiosis illustrates how regulated splicing improves yeast fitness. DNA breakage and recombination could be toxic during vegetative growth, and therefore entrance into meiosis is tightly controlled. Consequently, all 13 meiosis-specific introncontaining genes are regulated post-transcriptionally with splicing repressed during vegetative growth and induced during sporulation (Engebrecht et al. 1991;Nakagawa and Ogawa 1999;Juneau et al. 2007). Other examples of regulated splicing include YRA1 and MTR2 (RNA export) (Preker et al. 2002;Parenteau et al. 2008) and RPL30 (ribosomal) (Li et al. 1996).In contrast to regulated splicing, there is only one example in S. cerevisiae where splicing results in the expression of multiple proteins, SRC1 (Grund et al. 2008). SRC1 contains a single intron located at the 39 end of the pre-mRNA. The intron has two alternative 59-splice sites (Rodriguez-Navarro et al. 2002). Splicing at the most highly conserved 59-splice site (Bon et al. 2003) ...

Objective: To develop a microarray-based method for noninvasive prenatal testing (NIPT) and compare it with next-generation sequencing. Methods: Maternal plasma from 878 pregnant women, including 187 trisomy cases (18 trisomy 13, 37 trisomy 18, 132 trisomy 21), was evaluated for trisomy risk. Targeted chromosomes were analyzed using Digital Analysis of Selected Regions (DANSR™) assays. DANSR products were subsequently divided between two DNA quantification methods: microarrays and next-generation sequencing. For both microarray and sequencing methodologies, the Fetal-Fraction Optimized Risk of Trisomy Evaluation (FORTE™) algorithm was used to determine trisomy risk, assay variability across samples, and compute fetal fraction variability within samples. Results: NIPT using microarrays provided faster and more accurate cell-free DNA (cfDNA) measurements than sequencing. The assay variability, a measure of variance of chromosomal cfDNA counts, was lower for microarrays than for sequencing, 0.051 versus 0.099 (p < 0.0001). Analysis time using microarrays was faster, 7.5 versus 56 h for sequencing. Additionally, fetal fraction precision was improved 1.6-fold by assaying more polymorphic sites with microarrays (p < 0.0001). Microarrays correctly classified all trisomy and nontrisomy cases. Conclusions: NIPT using microarrays delivers more accurate cfDNA analysis than next-generation sequencing and can be performed in less time.