Meta-analysis of RNA-seq expression data across species, tissues and studies.

BackgroundDifferences in gene expression drive phenotypic differences between species, yet major organs and tissues generally have conserved gene expression programs. Several comparative transcriptomic studies have observed greater similarity in gene expression between homologous tissues from different vertebrate species than between diverse tissues of the same species. However, a recent study by Lin and colleagues reached the opposite conclusion. These studies differed in the species and tissues analyzed, and in technical details of library preparation, sequencing, read mapping, normalization, gene sets, and clustering methods.ResultsTo better understand gene expression evolution we reanalyzed data from four studies, including that of Lin, encompassing 6-13 tissues each from 11 vertebrate species using standardized mapping, normalization, and clustering methods. An analysis of independent data showed that the set of tissues chosen by Lin et al. were more similar to each other than those analyzed by previous studies. Comparing expression in five common tissues from the four studies, we observed that samples clustered exclusively by tissue rather than by species or study, supporting conservation of organ physiology in mammals. Furthermore, inter-study distances between homologous tissues were generally less than intra-study distances among different tissues, enabling informative meta-analyses. Notably, when comparing expression divergence of tissues over time to expression variation across 51 human GTEx tissues, we could accurately predict the clustering of expression for arbitrary pairs of tissues and species.ConclusionsThese results provide a framework for the design of future evolutionary studies of gene expression and demonstrate the utility of comparing RNA-seq data across studies

Interactome analysis brings splicing into focus

The spliceosome is a huge molecular machine that assembles dynamically onto its pre-mRNA substrates. A new study based on interactome analysis provides clues about how splicing-regulatory proteins modulate assembly of the spliceosome to either activate or repress splicing.National Cancer Institute (U.S.

Hollywood: a comparative relational database of alternative splicing

RNA splicing is an essential step in gene expression, and is often variable, giving rise to multiple alternatively spliced mRNA and protein isoforms from a single gene locus. The design of effective databases to support experimental and computational investigations of alternative splicing (AS) is a significant challenge. In an effort to integrate accurate exon and splice site annotation with current knowledge about splicing regulatory elements and predicted AS events, and to link information about the splicing of orthologous genes in different species, we have developed the Hollywood system. This database was built upon genomic annotation of splicing patterns of known genes derived from spliced alignment of complementary DNAs (cDNAs) and expressed sequence tags, and links features such as splice site sequence and strength, exonic splicing enhancers and silencers, conserved and non-conserved patterns of splicing, and cDNA library information for inferred alternative exons. Hollywood was implemented as a relational database and currently contains comprehensive information for human and mouse. It is accompanied by a web query tool that allows searches for sets of exons with specific splicing characteristics or splicing regulatory element composition, or gives a graphical or sequence-level summary of splicing patterns for a specific gene. A streamlined graphical representation of gene splicing patterns is provided, and these patterns can alternatively be layered onto existing information in the UCSC Genome Browser. The database is accessible at

Biased chromatin signatures around polyadenylation sites and exons

Core RNA-processing reactions in eukaryotic cells occur cotranscriptionally in a chromatin context, but the relationship between chromatin structure and pre-mRNA processing is poorly understood. We observed strong nucleosome depletion around human polyadenylation sites (PAS) and nucleosome enrichment just downstream of PAS. In genes with multiple alternative PAS, higher downstream nucleosome affinity was associated with higher PAS usage, independently of known PAS motifs that function at the RNA level. Conversely, exons were associated with distinct peaks in nucleosome density. Exons flanked by long introns or weak splice sites exhibited stronger nucleosome enrichment, and incorporation of nucleosome density data improved splicing simulation accuracy. Certain histone modifications, including H3K36me3 and H3K27me2, were specifically enriched on exons, suggesting active marking of exon locations at the chromatin level. Together, these findings provide evidence for extensive functional connections between chromatin structure and RNA processing

Widespread Inhibition of Posttranscriptional Splicing Shapes the Cellular Transcriptome following Heat Shock

During heat shock and other proteotoxic stresses, cells regulate multiple steps in gene expression in order to globally repress protein synthesis and selectively upregulate stress response proteins. Splicing of several mRNAs is known to be inhibited during heat stress, often meditated by SRp38, but the extent and specificity of this effect have remained unclear. Here, we examined splicing regulation genome-wide during heat shock in mouse fibroblasts. We observed widespread retention of introns in transcripts from ~1,700 genes, which were enriched for tRNA synthetase, nuclear pore, and spliceosome functions. Transcripts with retained introns were largely nuclear and untranslated. However, a group of 580+ genes biased for oxidation reduction and protein folding functions continued to be efficiently spliced. Interestingly, these unaffected transcripts are mostly cotranscriptionally spliced under both normal and stress conditions, whereas splicing-inhibited transcripts are mostly spliced posttranscriptionally. Altogether, our data demonstrate widespread repression of splicing in the mammalian heat stress response, disproportionately affecting posttranscriptionally spliced genes.Weizmann Institute of Science (Postdoctoral Award for Advancing Women in Science)European Molecular Biology Organization (Long-term Fellowship)Machiah FoundationNational Science Foundation (U.S.) (Grant 0821391)National Institutes of Health (U.S.

Numerous recursive sites contribute to accuracy of splicing of long introns in flies [preprint]

Recursive splicing, a process by which a single intron is removed from pre-mRNA transcripts in multiple distinct segments, has been observed in a small subset of Drosophila melanogaster introns. However, detection of recursive splicing requires observation of splicing intermediates which are inherently unstable, making it difficult to study. Here we developed new computational approaches to identify recursively spliced introns and applied them, in combination with existing methods, to nascent RNA sequencing data from Drosophila S2 cells. These approaches identified hundreds of novel sites of recursive splicing, expanding the catalog of recursively spliced fly introns by 4-fold. Recursive sites occur in most very long (\u3e 40 kb) fly introns, including many genes involved in morphogenesis and development, and tend to occur near the midpoints of introns. Suggesting a possible function for recursive splicing, we observe that fly introns with recursive sites are spliced more accurately than comparably sized non-recursive introns

Sashimi plots: Quantitative visualization of RNA sequencing read alignments

We introduce Sashimi plots, a quantitative multi-sample visualization of mRNA sequencing reads aligned to gene annotations. Sashimi plots are made using alignments (stored in the SAM/BAM format) and gene model annotations (in GFF format), which can be custom-made by the user or obtained from databases such as Ensembl or UCSC. We describe two implementations of Sashimi plots: (1) a stand-alone command line implementation aimed at making customizable publication quality figures, and (2) an implementation built into the Integrated Genome Viewer (IGV) browser, which enables rapid and dynamic creation of Sashimi plots for any genomic region of interest, suitable for exploratory analysis of alternatively spliced regions of the transcriptome. Isoform expression estimates outputted by the MISO program can be optionally plotted along with Sashimi plots. Sashimi plots can be used to quickly screen differentially spliced exons along genomic regions of interest and can be used in publication quality figures. The Sashimi plot software and documentation is available from: http://genes.mit.edu/burgelab/miso/docs/sashimi.htmlComment: 2 figure

A Combinatorial Code for Splicing Silencing: UAGG and GGGG Motifs

Alternative pre-mRNA splicing is widely used to regulate gene expression by tuning the levels of tissue-specific mRNA isoforms. Few regulatory mechanisms are understood at the level of combinatorial control despite numerous sequences, distinct from splice sites, that have been shown to play roles in splicing enhancement or silencing. Here we use molecular approaches to identify a ternary combination of exonic UAGG and 5′-splice-site-proximal GGGG motifs that functions cooperatively to silence the brain-region-specific CI cassette exon (exon 19) of the glutamate NMDA R1 receptor (GRIN1) transcript. Disruption of three components of the motif pattern converted the CI cassette into a constitutive exon, while predominant skipping was conferred when the same components were introduced, de novo, into a heterologous constitutive exon. Predominant exon silencing was directed by the motif pattern in the presence of six competing exonic splicing enhancers, and this effect was retained after systematically repositioning the two exonic UAGGs within the CI cassette. In this system, hnRNP A1 was shown to mediate silencing while hnRNP H antagonized silencing. Genome-wide computational analysis combined with RT-PCR testing showed that a class of skipped human and mouse exons can be identified by searches that preserve the sequence and spatial configuration of the UAGG and GGGG motifs. This analysis suggests that the multi-component silencing code may play an important role in the tissue-specific regulation of the CI cassette exon, and that it may serve more generally as a molecular language to allow for intricate adjustments and the coordination of splicing patterns from different genes