Conserved functions of lncRNAs during vertebrate development
Dr Alena Shkumatava, Institut Curie, France
Thousands of long intervening noncoding RNAs (lincRNAs) have been identified in mammals. To better understand functions and evolution of these enigmatic RNAs, we identified more than 550 lincRNAs in zebrafish, an established vertebrate model for development. Although zebrafish lincRNAs share many characteristics with mammalian lincRNAs, only 5% have detectable sequence similarity with putative mammalian counterparts, typically restricted to short regions of high conservation. To understand if evolutionarily pressure on conserved lincRNA sequences is associated with their important biological functions, Dr Shkumatava is generating multiple genetic zebrafish mutants of the ultra-conserved lincRNA motifs using Crispr/Cas9 genome editing. They are subsequently analyzing the impact of lincRNA loss-of-function on normal embryonic development and adult animals. For one of the ultra-conserved lincRNAs that they called cyrano, they recognized that the conserved region of cyrano contains an unusually complementary, near-perfect microRNA miR-7 site that is bound by Argonaute proteins and is highly conserved in all examined vertebrates. The analyses of zebrafish cyrano mutant show that the interaction between miR-7 and cyrano differs from the canonical miRNA—target regulation suggesting that lincRNA—miRNA complex has an additional, novel function important for normal development. To identify the exact molecular function of the cyrano–miR-7 pairing, they aim to identify proteins binding at the extended conserved region of Cyrano using a novel high-throughput method.
The nature and scaling of the regulatory superstructure of human development and cognition
Professor John Mattick, The Garvan Institute of Medical Research, Australia
It is now evident that the majority of the mammalian genome is dynamically transcribed during differentiation and development to produce tens if not hundreds of thousands of short and long non-protein-coding RNAs that show highly specific expression patterns and subcellular locations. Increasing numbers of these RNAs are being shown to have functions at many different levels of gene expression, including translational control and the guidance of epigenetic processes that underpin development, physiological adaptation, cognition and transgenerational communication, augmented by the superimposition of plasticity by RNA editing, RNA modification and retrotransposon mobilization. This suggests that there is there is a massive hidden layer of RNA-based communication and that the simple protein-centric operator-repressor model of ‘gene regulation’ derived from studies of bacteria is incorrect in highly organized and spatially specialized multicellular entities. This in turn requires reassessment of the nature, scaling and hierarchies of the regulatory systems and processes that control the 4-dimensional assembly and cognitive capacities of complex organisms. It is not simply a matter of adding RNA to the picture but rather of constructing a new landscape.
Transposable elements modulate human RNA abundance and splicing via specific RNA-protein interactions
Professor John Rinn, Harvard University, USA
Transposable elements (TEs) have significantly influenced the evolution of transcriptional regulatory networks in the human genome. Post-transcriptional regulation of human genes by TE-derived sequences has been observed in specific contexts, but has yet to be systematically and comprehensively investigated. Here, they study a collection of 75 CLIP-Seq experiments mapping the RNA binding sites for a diverse set of 51 human proteins to explore the role of TEs in post-transcriptional regulation of human mRNAs and lncRNAs via RNA-protein interactions.
Long non-coding RNAs that regulate hematopoiesis and adipogenesis
Professor Harvey Lodish, Massachusetts Institute of Technology, USA
To obtain a comprehensive view of how lncRNAs contribute to erythropoiesis, the Lodish lab performed and analyzed data from high depth RNA-sequencing on RNAs from erythroid progenitor cells and terminally differentiating erythroblasts. They focused on differentiation-induced lncRNAs, including novel erythroid-specific lncRNAs conserved in humans that are nuclear-localized and identified 13 erythroid-specific lncRNAs that are greatly induced during erythroid terminal differentiation. Importantly, shRNA-mediated loss-of-function assays reveal that all 13 are important for red cell formation. One intergenic lncRNA, LincRNA-EPS, prevents the apoptosis of progenitors that is normally induced by erythropoietin deprivation and represses expression of several proapoptotic genes including Pycard, a caspase activator. A second lncRNA is transcribed by the erythroid- specific enhancer of Band 3, encoding a major erythrocyte membrane protein. To uncover brown adipose tissue (BAT)-specific long non-coding RNAs (lncRNAs), they used high depth RNA-sequencing on RNAs from mouse brown, inguinal white, and epididymal white fat. They identified ~1500 lncRNAs, including 127 BAT-restricted loci induced during differentiation and often targeted by key regulators PPARγ, C/EBPα and C/EBPβ. One of them, lnc-BATE1, is required for establishment and maintenance of BAT identity and thermogenic capacity. lnc-BATE1 inhibition impairs concurrent activation of brown fat and repression of white fat genes, and is partially rescued by exogenous lnc-BATE1 with mutated siRNA-targeting sites, demonstrating a trans function of lnc-BATE1. Thus diverse types of intergenic, enhancer, and antisense lncRNAs are expressed only in specific types of hematopoietic and adipose cells and are essential for their proper development; they participate in the regulatory circuitry underlying lineage-specific development.
The theory of RNA-mediated gene evolution
Professor Kevin Morris, University of New South Wales, Australia
Observations over the last decade suggest that some non-coding RNAs function in regulating the transcriptional and epigenetic state of gene expression. DNA methylation appears operative in non-coding RNA regulation of gene expression. Interestingly, methylated cytosines undergo deamination to remove the methylation, which if not properly repaired results in the methylated cytosine being recognized by the cell as a thymine. Professor Morris finds that several of the key deamination based repair enzymes, APOBEC3a, Methyl-CpG (mCpG) binding domain protein 4 (MBD4), SMUG, and Uracil N-Glycosylase (UNG) localize to small and long non-coding RNA target loci. These observations suggest that the process of RNA directed epigenetic targeting also has the potential to alter the genomic landscape of the cell by changing cytosines to thymines and ultimately influence the evolution of the cell. This proposed theory of “RNA-mediated gene evolution” might be one possible mechanism of action whereby RNA participates in the natural selective process to drive cellular and possibly organismal evolution.