A high-resolution anatomical atlas of the transcriptome in the mouse embryo.

Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease.

A global view of gene activity and alternative splicing by deep sequencing of the human transcriptome.

The functional complexity of the human transcriptome is not yet fully elucidated. We report a high-throughput sequence of the human transcriptome from a human embryonic kidney and a B cell line. We used shotgun sequencing of transcripts to generate randomly distributed reads. Of these, 50% mapped to unique genomic locations, of which 80% corresponded to known exons. We found that 66% of the polyadenylated transcriptome mapped to known genes and 34% to nonannotated genomic regions. On the basis of known transcripts, RNA-Seq can detect 25% more genes than can microarrays. A global survey of messenger RNA splicing events identified 94,241 splice junctions (4096 of which were previously unidentified) and showed that exon skipping is the most prevalent form of alternative splicing.

Different levels of alternative splicing among eukaryotes.

Alternative splicing increases transcriptome and proteome diversification. Previous analyses aiming at comparing the rate of alternative splicing between different organisms provided contradicting results. These contradicting results were attributed to the fact that both analyses were dependent on the expressed sequence tag (EST) coverage, which varies greatly between the tested organisms. In this study we compare the level of alternative splicing among eight different organisms. By employing an EST independent approach we reveal that the percentage of genes and exons undergoing alternative splicing is higher in vertebrates compared with invertebrates. We also find that alternative exons of the skipping type are flanked by longer introns compared to constitutive ones, whereas alternative 5' and 3' splice sites events are generally not. In addition, although the regulation of alternative splicing and sizes of introns and exons have changed during metazoan evolution, intron retention remained the rarest type of alternative splicing, whereas exon skipping is more prevalent and exhibits a slight increase, from invertebrates to vertebrates. The difference in the level of alternative splicing suggests that alternative splicing may contribute greatly to the mammal higher level of phenotypic complexity, and that accumulation of introns confers an evolutionary advantage as it allows increasing the number of alternative splicing forms.

The importance of being divisible by three in alternative splicing.

Alternative splicing events that are conserved in orthologous genes in different species are commonly viewed as reliable evidence of authentic, functionally significant alternative splicing events. Several recent bioinformatic analyses have shown that conserved alternative exons possess several features that distinguish them from alternative exons that are species-specific. One of the most striking differences between conserved and species-specific alternative exons is the high percentage of exons that preserve the reading frame (exons whose length is an exact multiple of 3, termed symmetrical exons) among the conserved alternative exons. Here, we examined conserved alternative exons and found several features that differentiate between symmetrical and non-symmetrical alternative exons. We show that symmetrical alternative exons have a strong tendency not to disrupt protein domain structures, whereas the tendency of non-symmetrical alternative exons to overlap with different fractions of protein domains is similar to that of constitutive exons. Additionally, skipping isoforms of non-symmetrical alternative exons are strongly underrepresented, compared with their including isoforms, suggesting that skipping of a large fraction of non-symmetrical alternative exons produces transcripts that are degraded by the nonsense-mediated mRNA decay mechanism. Non-symmetrical alternative exons also show a tendency to reside in the 5' half of the CDS. These findings suggest that alternative splicing of symmetrical and non-symmetrical exons is governed by different selective pressures and serves different purposes.