Circular RNA repertoires are associated with evolutionarily young transposable elements.

Circular RNAs (circRNAs) are found across eukaryotes and can function in post-transcriptional gene regulation. Their biogenesis through a circle-forming backsplicing reaction is facilitated by reverse-complementary repetitive sequences promoting pre-mRNA folding. Orthologous genes from which circRNAs arise, overall contain more strongly conserved splice sites and exons than other genes, yet it remains unclear to what extent this conservation reflects purifying selection acting on the circRNAs themselves. Our analyses of circRNA repertoires from five species representing three mammalian lineages (marsupials, eutherians: rodents, primates) reveal that surprisingly few circRNAs arise from orthologous exonic loci across all species. Even the circRNAs from orthologous loci are associated with young, recently active and species-specific transposable elements, rather than with common, ancient transposon integration events. These observations suggest that many circRNAs emerged convergently during evolution - as a byproduct of splicing in orthologs prone to transposon insertion. Overall, our findings argue against widespread functional circRNA conservation.

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SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

ROP: dumpster diving in RNA-sequencing to find the source of 1 trillion reads across diverse adult human tissues.

High-throughput RNA-sequencing (RNA-seq) technologies provide an unprecedented opportunity to explore the individual transcriptome. Unmapped reads are a large and often overlooked output of standard RNA-seq analyses. Here, we present Read Origin Protocol (ROP), a tool for discovering the source of all reads originating from complex RNA molecules. We apply ROP to samples across 2630 individuals from 54 diverse human tissues. Our approach can account for 99.9% of 1 trillion reads of various read length. Additionally, we use ROP to investigate the functional mechanisms underlying connections between the immune system, microbiome, and disease. ROP is freely available at https://github.com/smangul1/rop/wiki .

High resolution temporal transcriptomics of mouse embryoid body development reveals complex expression dynamics of coding and noncoding loci.

Cellular responses to stimuli are rapid and continuous and yet the vast majority of investigations of transcriptional responses during developmental transitions typically use long interval time courses; limiting the available interpretive power. Moreover, such experiments typically focus on protein-coding transcripts, ignoring the important impact of long noncoding RNAs. We therefore evaluated coding and noncoding expression dynamics at unprecedented temporal resolution (6-hourly) in differentiating mouse embryonic stem cells and report new insight into molecular processes and genome organization. We present a highly resolved differentiation cascade that exhibits coding and noncoding transcriptional alterations, transcription factor network interactions and alternative splicing events, little of which can be resolved by long-interval developmental time-courses. We describe novel short lived and cycling patterns of gene expression and dissect temporally ordered gene expression changes in response to transcription factors. We elucidate patterns in gene co-expression across the genome, describe asynchronous transcription at bidirectional promoters and functionally annotate known and novel regulatory lncRNAs. These findings highlight the complex and dynamic molecular events underlying mammalian differentiation that can only be observed though a temporally resolved time course.

Identification, visualization and clonal analysis of intestinal stem cells in fish.

Recently, a stochastic model of symmetrical stem cell division followed by neutral drift has been proposed for intestinal stem cells (ISCs), which has been suggested to represent the predominant mode of stem cell progression in mammals. In contrast, stem cells in the retina of teleost fish show an asymmetric division mode. To address whether the mode of stem cell division follows phylogenetic or ontogenetic routes, we analysed the entire gastrointestinal tract of the teleost medaka (Oryzias latipes). X-ray microcomputed tomography shows a correlation of 3D topography with the functional domains. Analysis of ISCs in proliferation assays and via genetically encoded lineage tracing highlights a stem cell niche in the furrow between the long intestinal folds that is functionally equivalent to mammalian intestinal crypts. Stem cells in this compartment are characterized by the expression of homologs of mammalian ISC markers - sox9, axin2 and lgr5 - emphasizing the evolutionary conservation of the Wnt pathway components in the stem cell niche of the intestine. The stochastic, sparse initial labelling of ISCs ultimately resulted in extended labelled or unlabelled domains originating from single stem cells in the furrow niche, contributing to both homeostasis and growth. Thus, different modes of stem cell division co-evolved within one organism, and in the absence of physical isolation in crypts, ISCs contribute to homeostatic growth.

The Evx1/Evx1as gene locus regulates anterior-posterior patterning during gastrulation.

Thousands of sense-antisense mRNA-lncRNA gene pairs occur in the mammalian genome. While there is usually little doubt about the function of the coding transcript, the function of the lncRNA partner is mostly untested. Here we examine the function of the homeotic Evx1-Evx1as gene locus. Expression is tightly co-regulated in posterior mesoderm of mouse embryos and in embryoid bodies. Expression of both genes is enhanced by BMP4 and WNT3A, and reduced by Activin. We generated a suite of deletions in the locus by CRISPR-Cas9 editing. We show EVX1 is a critical downstream effector of BMP4 and WNT3A with respect to patterning of posterior mesoderm. The lncRNA, Evx1as arises from alternative promoters and is difficult to fully abrogate by gene editing or siRNA approaches. Nevertheless, we were able to generate a large 2.6 kb deletion encompassing the shared promoter with Evx1 and multiple additional exons of Evx1as. This led to an identical dorsal-ventral patterning defect to that generated by micro-deletion in the DNA-binding domain of EVX1. Thus, Evx1as has no function independent of EVX1, and is therefore unlikely to act in trans. We predict many antisense lncRNAs have no specific trans function, possibly only regulating the linked coding genes in cis.

Combining computational prediction of cis-regulatory elements with a new enhancer assay to efficiently label neuronal structures in the medaka fish.

The developing vertebrate nervous system contains a remarkable array of neural cells organized into complex, evolutionarily conserved structures. The labeling of living cells in these structures is key for the understanding of brain development and function, yet the generation of stable lines expressing reporter genes in specific spatio-temporal patterns remains a limiting step. In this study we present a fast and reliable pipeline to efficiently generate a set of stable lines expressing a reporter gene in multiple neuronal structures in the developing nervous system in medaka. The pipeline combines both the accurate computational genome-wide prediction of neuronal specific cis-regulatory modules (CRMs) and a newly developed experimental setup to rapidly obtain transgenic lines in a cost-effective and highly reproducible manner. 95% of the CRMs tested in our experimental setup show enhancer activity in various and numerous neuronal structures belonging to all major brain subdivisions. This pipeline represents a significant step towards the dissection of embryonic neuronal development in vertebrates.