Ian Bell

RNA Maps Reveal New RNA Classes and a Possible Function for Pervasive Transcription

Significant fractions of eukaryotic genomes give rise to RNA, much of which is unannotated and has reduced protein-coding potential. The genomic origins and the associations of human nuclear and cytosolic polyadenylated RNAs longer than 200 nucleotides (nt) and whole-cell RNAs less than 200 nt were investigated in this genome-wide study. Subcellular addresses for nucleotides present in detected RNAs were assigned, and their potential processing into short RNAs was investigated. Taken together, these observations suggest a novel role for some unannotated RNAs as primary transcripts for the production of short RNAs. Three potentially functional classes of RNAs have been identified, two of which are syntenically conserved and correlate with the expression state of protein-coding genes. These data support a highly interleaved organization of the human transcriptome.

Evidence for Transcript Networks Composed of Chimeric RNAs in Human Cells

The classic organization of a gene structure has followed the Jacob and Monod bacterial gene model proposed more than 50 years ago. Since then, empirical determinations of the complexity of the transcriptomes found in yeast to human has blurred the definition and physical boundaries of genes. Using multiple analysis approaches we have characterized individual gene boundaries mapping on human chromosomes 21 and 22. Analyses of the locations of the 5′ and 3′ transcriptional termini of 492 protein coding genes revealed that for 85% of these genes the boundaries extend beyond the current annotated termini, most often connecting with exons of transcripts from other well annotated genes. The biological and evolutionary importance of these chimeric transcripts is underscored by (1) the non-random interconnections of genes involved, (2) the greater phylogenetic depth of the genes involved in many chimeric interactions, (3) the coordination of the expression of connected genes and (4) the close in vivo and three dimensional proximity of the genomic regions being transcribed and contributing to parts of the chimeric RNAs. The non-random nature of the connection of the genes involved suggest that chimeric transcripts should not be studied in isolation, but together, as an RNA network.

Biological function of unannotated transcription during the early development of Drosophila melanogaster

Many animal and plant genomes are transcribed much more extensively than current annotations predict. However, the biological function of these unannotated transcribed regions is largely unknown. Approximately 7% and 23% of the detected transcribed nucleotides during D. melanogaster embryogenesis map to unannotated intergenic and intronic regions, respectively. Based on computational analysis of coordinated transcription, we conservatively estimate that 29% of all unannotated transcribed sequences function as missed or alternative exons of well-characterized protein-coding genes. We estimate that 15.6% of intergenic transcribed regions function as missed or alternative transcription start sites (TSS) used by 11.4% of the expressed protein-coding genes. Identification of P element mutations within or near newly identified 5′ exons provides a strategy for mapping previously uncharacterized mutations to their respective genes. Collectively, these data indicate that at least 85% of the fly genome is transcribed and processed into mature transcripts representing at least 30% of the fly genome.

Linking promoters to functional transcripts in small samples with nanoCAGE and CAGEscan

Large-scale sequencing projects have revealed an unexpected complexity in the origins, structures and functions of mammalian transcripts. Many loci are known to produce overlapping coding and noncoding RNAs with capped 5′ ends that vary in size. Methods to identify the 5′ ends of transcripts will facilitate the discovery of new promoters and 5′ ends derived from secondary capping events. Such methods often require high input amounts of RNA not obtainable from highly refined samples such as tissue microdissections and subcellular fractions. Therefore, we developed nano–cap analysis of gene expression (nanoCAGE), a method that captures the 5′ ends of transcripts from as little as 10 ng of total RNA, and CAGEscan, a mate-pair adaptation of nanoCAGE that captures the transcript 5′ ends linked to a downstream region. Both of these methods allow further annotation-agnostic studies of the complex human transcriptome.

Stability and Dynamics of Polycomb Target Sites in Drosophila Development

Polycomb-group (PcG) and Trithorax-group proteins together form a maintenance machinery that is responsible for stable heritable states of gene activity. While the best-studied target genes are the Hox genes of the Antennapedia and Bithorax complexes, a large number of key developmental genes are also Polycomb (Pc) targets, indicating a widespread role for this maintenance machinery in cell fate determination. We have studied the linkage between the binding of PcG proteins and the developmental regulation of gene expression using whole-genome mapping to identify sites bound by the PcG proteins, Pc and Pleiohomeotic (Pho), in the Drosophila embryo and in a more restricted tissue, the imaginal discs of the third thoracic segment. Our data provide support for the idea that Pho is a general component of the maintenance machinery, since the majority of Pc targets are also associated with Pho binding. We find, in general, considerable developmental stability of Pc and Pho binding at target genes and observe that Pc/Pho binding can be associated with both expressed and inactive genes. In particular, at the Hox complexes, both active and inactive genes have significant Pc and Pho binding. However, in comparison to inactive genes, the active Hox genes show reduced and altered binding profiles. During development, Pc target genes are not simply constantly associated with Pc/Pho binding, and we identify sets of genes with clear differential binding between embryo and imaginal disc. Using existing datasets, we show that for specific fate-determining genes of the haemocyte lineage, the active state is characterised by lack of Pc binding. Overall, our analysis suggests a dynamic relationship between Pc/Pho binding and gene transcription. Pc/Pho binding does not preclude transcription, but levels of Pc/Pho binding change during development, and loss of Pc/Pho binding can be associated with both stable gene activity and inactivity.

High resolution transcriptome maps for wild-type and nonsense-mediated decay-defective Caenorhabditis elegans

BackgroundWhile many genome sequences are complete, transcriptomes are less well characterized. We used both genome-scale tiling arrays and massively parallel sequencing to map the Caenorhabditis elegans transcriptome across development. We utilized this framework to identify transcriptome changes in animals lacking the nonsense-mediated decay (NMD) pathway.ResultsWe find that while the majority of detectable transcripts map to known gene structures, >5% of transcribed regions fall outside current gene annotations. We show that >40% of these are novel exons. Using both technologies to assess isoform complexity, we estimate that >17% of genes change isoform across development. Next we examined how the transcriptome is perturbed in animals lacking NMD. NMD prevents expression of truncated proteins by degrading transcripts containing premature termination codons. We find that approximately 20% of genes produce transcripts that appear to be NMD targets. While most of these arise from splicing errors, NMD targets are enriched for transcripts containing open reading frames upstream of the predicted translational start (uORFs). We identify a relationship between the Kozak consensus surrounding the true start codon and the degree to which uORF-containing transcripts are targeted by NMD and speculate that translational efficiency may be coupled to transcript turnover via the NMD pathway for some transcripts.ConclusionsWe generated a high-resolution transcriptome map for C. elegans and used it to identify endogenous targets of NMD. We find that these transcripts arise principally through splicing errors, strengthening the prevailing view that splicing and NMD are highly interlinked processes.

Comprehensive identification of Drosophila dorsal-ventral patterning genes using a whole-genome tiling array

Dorsal–ventral (DV) patterning of the Drosophila embryo is initiated by Dorsal, a sequence-specific transcription factor distributed in a broad nuclear gradient in the precellular embryo. Previous studies have identified as many as 70 protein-coding genes and one microRNA (miRNA) gene that are directly or indirectly regulated by this gradient. A gene regulation network, or circuit diagram, including the functional interconnections among 40 Dorsal target genes and 20 associated tissue-specific enhancers, has been determined for the initial stages of gastrulation. Here, we attempt to extend this analysis by identifying additional DV patterning genes using a recently developed whole-genome tiling array. This analysis led to the identification of another 30 protein-coding genes, including the Drosophila homolog of Idax, an inhibitor of Wnt signaling. In addition, remote 5′ exons were identified for at least 10 of the ≈100 protein-coding genes that were missed in earlier annotations. As many as nine intergenic uncharacterized transcription units were identified, including two that contain known microRNAs, miR-1 and -9a. We discuss the potential functions of these recently identified genes and suggest that intronic enhancers are a common feature of the DV gene network.

A Comparison of the Seasonal Movements of Tiger Sharks and Green Turtles Provides Insight into Their Predator-Prey Relationship

During the reproductive season, sea turtles use a restricted area in the vicinity of their nesting beaches, making them vulnerable to predation. At Raine Island (Australia), the highest density green turtle Chelonia mydas rookery in the world, tiger sharks Galeocerdo cuvier have been observed to feed on green turtles, and it has been suggested that they may specialise on such air-breathing prey. However there is little information with which to examine this hypothesis. We compared the spatial and temporal components of movement behaviour of these two potentially interacting species in order to provide insight into the predator-prey relationship. Specifically, we tested the hypothesis that tiger shark movements are more concentrated at Raine Island during the green turtle nesting season than outside the turtle nesting season when turtles are not concentrated at Raine Island. Turtles showed area-restricted search behaviour around Raine Island for ∼3–4 months during the nesting period (November–February). This was followed by direct movement (transit) to putative foraging grounds mostly in the Torres Straight where they switched to area-restricted search mode again, and remained resident for the remainder of the deployment (53–304 days). In contrast, tiger sharks displayed high spatial and temporal variation in movement behaviour which was not closely linked to the movement behaviour of green turtles or recognised turtle foraging grounds. On average, tiger sharks were concentrated around Raine Island throughout the year. While information on diet is required to determine whether tiger sharks are turtle specialists our results support the hypothesis that they target this predictable and plentiful prey during turtle nesting season, but they might not focus on this less predictable food source outside the nesting season.

Behavioral evidence suggests facultative scavenging by a marine apex predator during a food pulse

The ability of predators to switch between hunting and scavenging (facultative scavenging) carries both short-term survival and long-term fitness advantages. However, the mechanistic basis for facultative scavenging remains poorly understood. The co-occurrence of tiger sharks (Galeocerdo cuvier) and green turtles (Chelonia mydas) at Raine Island (Australia), provides an opportunity to examine a top marine predator’s feeding mode in response to seasonal pulses in nesting turtles that offer both hunting and scavenging opportunities. Using satellite telemetry, we evaluated home range overlap between sharks and turtles and quantified their surfacing behavior around Raine Island during the turtle nesting season. We found core home range overlap to be highest during the nesting season. Both sharks and turtles spent significantly more time at the surface in areas of greatest range overlap closest to shore, where turtle density was highest. Both sharks and turtles showed decreased surfacing with increasing distance from Raine Island. Combined with published data on turtle demography at Raine Island, we propose the following: (1) sharks patrol the surface to increase scavenging opportunities on turtle carcasses and intercept weakened individuals after nesting; (2) healthy turtles may not perceive sharks as a major threat and/or other biological factors override anti-predatory responses; and (3) sharks during the nesting season may primarily scavenge on dead turtles individuals rather than actively hunt. Our study results and approach may be applicable to other situations in which direct observations of predator-prey interactions are limited.Significance StatementEvery animal encounters dead or dying resources, yet the role of facultative scavenging has been difficult to study, and thus largely overlooked in marine behavioral ecological research. Movement analyses of tiger shark and green turtle movement and surfacing behavior at Raine Island (Australia) suggest that facultative scavenging may be a prevalent, yet underappreciated, feeding strategy in tiger sharks. Our integration of behavioral ecology theory with multi-species electronic tagging provided a valuable approach for investigating predator-prey interactions in situations where direct observations are limited or not possible.