Gemma May

Diversity and dynamics of the Drosophila transcriptome

Animal transcriptomes are dynamic, with each cell type, tissue and organ system expressing an ensemble of transcript isoforms that give rise to substantial diversity. Here we have identified new genes, transcripts and proteins using poly(A)+ RNA sequencing from Drosophila melanogaster in cultured cell lines, dissected organ systems and under environmental perturbations. We found that a small set of mostly neural-specific genes has the potential to encode thousands of transcripts each through extensive alternative promoter usage and RNA splicing. The magnitudes of splicing changes are larger between tissues than between developmental stages, and most sex-specific splicing is gonad-specific. Gonads express hundreds of previously unknown coding and long non-coding RNAs (lncRNAs), some of which are antisense to protein-coding genes and produce short regulatory RNAs. Furthermore, previously identified pervasive intergenic transcription occurs primarily within newly identified introns. The fly transcriptome is substantially more complex than previously recognized, with this complexity arising from combinatorial usage of promoters, splice sites and polyadenylation sites.

Transposon-mediated rewiring of gene regulatory networks contributed to the evolution of pregnancy in mammals

A fundamental challenge in biology is explaining the origin of novel phenotypic characters such as new cell types; the molecular mechanisms that give rise to novelties are unclear. We explored the gene regulatory landscape of mammalian endometrial cells using comparative RNA-Seq and found that 1,532 genes were recruited into endometrial expression in placental mammals, indicating that the evolution of pregnancy was associated with a large-scale rewiring of the gene regulatory network. About 13% of recruited genes are within 200 kb of a Eutherian-specific transposable element (MER20). These transposons have the epigenetic signatures of enhancers, insulators and repressors, directly bind transcription factors essential for pregnancy and coordinately regulate gene expression in response to progesterone and cAMP. We conclude that the transposable element, MER20, contributed to the origin of a novel gene regulatory network dedicated to pregnancy in placental mammals, particularly by recruiting the cAMP signaling pathway into endometrial stromal cells.

Comparative analysis of the transcriptome across distant species.

The transcriptome is the readout of the genome. Identifying common features in it across distant species can reveal fundamental principles. To this end, the ENCODE and modENCODE consortia have generated large amounts of matched RNA-sequencing data for human, worm and fly. Uniform processing and comprehensive annotation of these data allow comparison across metazoan phyla, extending beyond earlier within-phylum transcriptome comparisons and revealing ancient, conserved features. Specifically, we discover co-expression modules shared across animals, many of which are enriched in developmental genes. Moreover, we use expression patterns to align the stages in worm and fly development and find a novel pairing between worm embryo and fly pupae, in addition to the embryo-to-embryo and larvae-to-larvae pairings. Furthermore, we find that the extent of non-canonical, non-coding transcription is similar in each organism, per base pair. Finally, we find in all three organisms that the gene-expression levels, both coding and non-coding, can be quantitatively predicted from chromatin features at the promoter using a ‘universal model’ based on a single set of organism-independent parameters.

Mod-seq: high-throughput sequencing for chemical probing of RNA structure

The functions of RNA molecules are intimately linked to their ability to fold into complex secondary and tertiary structures. Thus, understanding how these molecules fold is essential to determining how they function. Current methods for investigating RNA structure often use small molecules, enzymes, or ions that cleave or modify the RNA in a solvent-accessible manner. While these methods have been invaluable to understanding RNA structure, they can be fairly labor intensive and often focus on short regions of single RNAs. Here we present a new method (Mod-seq) and data analysis pipeline (Mod-seeker) for assaying the structure of RNAs by high-throughput sequencing. This technique can be utilized both in vivo and in vitro, with any small molecule that modifies RNA and consequently impedes reverse transcriptase. As proof-of-principle, we used dimethyl sulfate (DMS) to probe the in vivo structure of total cellular RNAs in Saccharomyces cerevisiae. Mod-seq analysis simultaneously revealed secondary structural information for all four ribosomal RNAs and 32 additional noncoding RNAs. We further show that Mod-seq can be used to detect structural changes in 5.8S and 25S rRNAs in the absence of ribosomal protein L26, correctly identifying its binding site on the ribosome. While this method is applicable to RNAs of any length, its high-throughput nature makes Mod-seq ideal for studying long RNAs and complex RNA mixtures.

Ribosome profiling reveals post-transcriptional buffering of divergent gene expression in yeast

Understanding the patterns and causes of phenotypic divergence is a central goal in evolutionary biology. Much work has shown that mRNA abundance is highly variable between closely related species. However, the extent and mechanisms of post-transcriptional gene regulatory evolution are largely unknown. Here we used ribosome profiling to compare transcript abundance and translation efficiency in two closely related yeast species (S. cerevisiae and S. paradoxus). By comparing translation regulatory divergence to interspecies differences in mRNA sequence features, we show that differences in transcript leaders and codon bias substantially contribute to divergent translation. Globally, we find that translation regulatory divergence often buffers species differences in mRNA abundance, such that ribosome occupancy is more conserved than transcript abundance. We used allele-specific ribosome profiling in interspecies hybrids to compare the relative contributions of cis- and trans-regulatory divergence to species differences in mRNA abundance and translation efficiency. The mode of gene regulatory divergence differs for these processes, as trans-regulatory changes play a greater role in divergent mRNA abundance than in divergent translation efficiency. Strikingly, most genes with aberrant transcript abundance in F1 hybrids (either over- or underexpressed compared to both parent species) did not exhibit aberrant ribosome occupancy. Our results show that interspecies differences in translation contribute substantially to the evolution of gene expression. Compensatory differences in transcript abundance and translation efficiency may increase the robustness of gene regulation.

Regulatory evolution through divergence of a phosphoswitch in the transcription factor CEBPB.

There is an emerging consensus that gene regulation evolves through changes in cis-regulatory elements and transcription factors. Although it is clear how nucleotide substitutions in cis-regulatory elements affect gene expression, it is not clear how amino-acid substitutions in transcription factors influence gene regulation. Here we show that amino-acid changes in the transcription factor CCAAT/enhancer binding protein-β (CEBPB, also known as C/EBP-β) in the stem-lineage of placental mammals changed the way it responds to cyclic AMP/protein kinase A (cAMP/PKA) signalling. By functionally analysing resurrected ancestral proteins, we identify three amino-acid substitutions in an internal regulatory domain of CEBPB that are responsible for the novel function. These amino-acid substitutions reorganize the location of key phosphorylation sites, introducing a new site and removing two ancestral sites, reversing the response of CEBPB to GSK-3β-mediated phosphorylation from repression to activation. We conclude that changing the response of transcription factors to signalling pathways can be an important mechanism of gene regulatory evolution.

The Genome of Anopheles darlingi , the main neotropical malaria vector

Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vector–human and vector–parasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles-darlingi.

DNA copy number evolution in Drosophila cell lines

BackgroundStructural rearrangements of the genome resulting in genic imbalance due to copy number change are often deleterious at the organismal level, but are common in immortalized cell lines and tumors, where they may be an advantage to cells. In order to explore the biological consequences of copy number changes in the Drosophila genome, we resequenced the genomes of 19 tissue-culture cell lines and generated RNA-Seq profiles.ResultsOur work revealed dramatic duplications and deletions in all cell lines. We found three lines of evidence indicating that copy number changes were due to selection during tissue culture. First, we found that copy numbers correlated to maintain stoichiometric balance in protein complexes and biochemical pathways, consistent with the gene balance hypothesis. Second, while most copy number changes were cell line-specific, we identified some copy number changes shared by many of the independent cell lines. These included dramatic recurrence of increased copy number of the PDGF/VEGF receptor, which is also over-expressed in many cancer cells, and of bantam, an anti-apoptosis miRNA. Third, even when copy number changes seemed distinct between lines, there was strong evidence that they supported a common phenotypic outcome. For example, we found that proto-oncogenes were over-represented in one cell line (S2-DRSC), whereas tumor suppressor genes were under-represented in another (Kc167).ConclusionOur study illustrates how genome structure changes may contribute to selection of cell lines in vitro. This has implications for other cell-level natural selection progressions, including tumorigenesis.

Competing RNA secondary structures are required for mutually exclusive splicing of the Dscam exon 6 cluster

Alternative splicing of eukaryotic pre-mRNAs is an important mechanism for generating proteome diversity and regulating gene expression. The Drosophila melanogaster Down Syndrome Cell Adhesion Molecule (Dscam) gene is an extreme example of mutually exclusive splicing. Dscam contains 95 alternatively spliced exons that potentially encode 38,016 distinct mRNA and protein isoforms. We previously identified two sets of conserved sequence elements, the docking site and selector sequences in the Dscam exon 6 cluster, which contains 48 mutually exclusive exons. These elements were proposed to engage in competing RNA secondary structures required for mutually exclusive splicing, though this model has not yet been experimentally tested. Here we describe a new system that allowed us to demonstrate that the docking site and selector sequences are indeed required for exon 6 mutually exclusive splicing and that the strength of these RNA structures determines the frequency of exon 6 inclusion. We also show that the function of the docking site has been conserved for ~500 million years of evolution. This work demonstrates that conserved intronic sequences play a functional role in mutually exclusive splicing of the Dscam exon 6 cluster.

Evolution of digit identity in the three-toed Italian skink Chalcides chalcides: a new case of digit identity frame shift

SUMMARY Digit identity in the avian wing is a classical example of conflicting anatomical and embryological evidence regarding digit homology. Anatomical in conjunction with phylogenetic evidence supports the hypothesis that the three remaining digits in the bird wing are digits 1, 2, and 3. At the same time, various lines of embryological evidence support the notion that these digits develop in positions that normally produce digits 2, 3, and 4. In recent years, gene expression as well as experimental evidence was published that supports the hypothesis that this discrepancy arose from a digit identity shift in the evolution of the bird wing. A similar but less well‐known controversy has been ongoing since the late 19th century regarding the identity of the digits of the three‐toed Italian skink, Chalcides chalcides. Comparative anatomy identifies these digits as 1, 2, and 3, while embryological evidence suggests their derivation from embryological positions 2, 3, and 4. Here we re‐examine this evidence and add gene expression data to determine the identity of the three digits of C. chalcides. The data confirm that the adult and the embryological evidence for digit identity are in conflict, and the expression of Hoxd11 suggests that digits 1, 2, and 3 develop in positions 2, 3, and 4. We conclude that in C. chalcides, and likely in its close relatives, a digit identity frame shift has occurred, similar to the one in avian evolution. This result suggests that changes in of digit identity might be a more frequent consequence of digit reduction than previously assumed.