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Long non-coding RNAs: evolution of new epigenetic and post-transcriptional functions

Event

Location

Kavli Royal Society Centre, Chicheley Hall, Newport Pagnell, Buckinghamshire, MK16 9JJ

Overview

Theo Murphy scientific meeting organised by Professor Leonard Lipovich, Professor John Rinn, Professor Douglas Higgs FRS, Professor Nicholas Proudfoot FRS and Professor Lynne Maquat

A pseudogene antisense lncRNA targets the PTEN promoter, interacting with nascent mRNA and modifying chromatin. Credit: Professor Kevin V. Morris, University of New South Wales, used by permission.

Event details

The discovery that long non-protein-coding RNAs (lncRNAs) are prevalent in metazoan transcriptomes has been a highlight of the second post-genomic decade. Unexpectedly, lncRNAs mediate remarkably diverse cellular mechanisms. This meeting will synthesise insights that derive from next-generation sequencing, functional genomics, and lncRNA structure and function to augment our understanding of how lncRNAs contribute to species uniqueness and to human disease.

Biographies of the speakers are available below. Recorded audio of the presentations will be available on this page after the event.

Attending this event

This is a residential conference, which allows for increased discussion and networking. It is free to attend, however participants need to cover their accommodation and catering costs if required.

Enquiries: Contact the events team

Event organisers

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Schedule of talks

28 September

09:00-12:30

Session 1: Genomic cartography of the lncRNA world

5 talks Show detail Hide detail

Chairs

Professor Lynne Maquat, University of Rochester Medical Centre, USA

09:05-09:30 Conserved functions of lncRNAs during vertebrate development

Dr Alena Shkumatava, Institut Curie, France

Abstract

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.

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09:30-09:55 The nature and scaling of the regulatory superstructure of human development and cognition

Professor John Mattick, The Garvan Institute of Medical Research, Australia

Abstract

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.

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10:45-11:10 Transposable elements modulate human RNA abundance and splicing via specific RNA-protein interactions

Professor John Rinn, Harvard University, USA

Abstract

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.

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11:10-11:35 Long non-coding RNAs that regulate hematopoiesis and adipogenesis

Professor Harvey Lodish, Massachusetts Institute of Technology, USA

Abstract

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.

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11:35-12:00 The theory of RNA-mediated gene evolution

Professor Kevin Morris, University of New South Wales, Australia

Abstract

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. 

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12:30-13:30 Lunch

13:30-17:00

Session 2: Nuclear and cytoplasmic lncRNA functions

4 talks Show detail Hide detail

Chairs

Professor John Rinn, Harvard University, USA

13:30-13:55 The origins and potential roles of non-coding RNAs in a well-defined mammalian chromosomal landscape

Dr Bryony Graham, University of Oxford, UK

Abstract

Gene expression in mammalian cells is a complex process which is often influenced by distal regulatory elements. The alpha globin gene locus, amongst others, has been thoroughly characterized as a model of such long-range gene regulation. Mutations which affect some or all of the distal regulatory elements in the human alpha globin gene locus result in alpha thalassemia, illustrating the relevance of long-range gene regulation in vivo. Recent studies have shown that distal regulatory elements (e.g. enhancers) physically interact with their target gene promoters and are themselves actively transcribed by RNA Pol II. Production of RNA transcripts from enhancer elements has been shown on a genome-wide scale in a number of different cell types, including terminally differentiated erythroid cells, but whether these transcripts have a biological function or merely represent transcriptional noise is not fully understood. Dr Graham has optimized a method to recapitulate mouse erythropoiesis in vitro, and use this system to characterize the transcriptional activity of enhancer elements through the differentiation of haematopoietic progenitors to terminally differentiated erythroid cells. Using genetic knockout models of individual regulatory elements within the well-defined alpha globin locus, she asks whether transcriptional activity at an enhancer reflects its ability to regulate activity at a downstream promoter in vivo.

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13:55-14:20 Policing the mammalian transcriptome: restricting non-coding RNAs by transcriptional termination, gene loops and R-loops

Professor Nicholas Proudfoot FRS, University of Oxford, UK

Abstract

The highly unstable nature of lncRNA requires their analysis by truly nascent transcription procedures. Professor Proudfoot has developed mammalian native elongation transcript sequencing (mNET-seq) analysis as a way to determine nascent transcription profiles and their association with specific phosphorylation states of RNA polymerase II CTD (Nojima et al Cell 161: 526, 2015). Their analysis to date has concentrated on the HeLa cell transcriptome. mNET-seq involves the isolation and sequencing of RNA from the Pol II active site and gives single nucleotide transcript resolution. Their results so far indicate that lncRNA and pre-mRNA are transcribed in significantly different ways. While pre-mRNA are associated with specific CTD phosphorylation states indicative of co-transcriptional splicing and 3’ end processing/polyadenylation, lncRNA are largely transcribed by different Pol II CTD isoforms. Furthermore most lncRNA are retained on chromatin where they are co-transcriptionally degraded. He further shows that some lncRNA initiate from single stranded DNA formed as a result of RNA:DNA hybrid formation; R-loop regions. Overall he shows that the transcriptional and RNA processing mechanisms that generate lncRNA appear radically different to pre-mRNA. Our results imply that lncRNA are in most cases transcriptional bi-products that need to be degraded in situ to prevent their deleterious effects on cell physiology.

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15:10-15:35 Epitranscriptome and transcriptome dynamics in single-cells, nanopores and space stations

Dr Christopher Mason, Weill Cornell Medical College, USA

Abstract

The avalanche of easy-to-create genomics data has impacted almost all areas of medicine and science, and here Dr Mason reports the implementation of genomics technologies from the single-cell to an entire city.  Recent methods and algorithms enable single-cell and clonal resolution of phenotypes as they evolve, both in normal and diseased tissues.  He shows that tumours evolve quickly at the genetic, epigenetic, transcriptional, and epitranscripional level, enabling many means by which tumours can resist therapy.  Notably, some of these changes can be resolved by single-cell analysis and enable prognostic relevance.  Finally, he will discuss pilot data for creating enabling patients to become more involved in their ‘omics data, including to an integrative genomics view of an entire city (based on our Pathomap project) that leverages longitudinal genomics and microbiome profiles of the NYC subway system and in situ sequencing with the oxford nanopore system of sequencing.   All of these pieces work together to guide the most comprehensive, longitudinal, mutli-omic view of human physiology with the NASA Twins Study.

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15:45-17:00 Poster session

29 September

09:00-12:30

Session 3: Structural biology of the lncRNAome

5 talks Show detail Hide detail

Chairs

Professor Douglas Higgs FRS, University of Oxford, UK

09:00-09:25 "Alu"strious effects on human RNA metabolism: a complicated network

Professor Lynne Maquat, University of Rochester Medical Centre, USA

Abstract

Staufen1-mediated mRNA decay (SMD), which occurs when translation terminates sufficiently upstream of a STAU-binding site (SBS), is important to developmental and homeostatic pathways. An SBS can be created by intramolecular base-pairing within an mRNA 3'UTR or by intermolecular base-pairing between a 3'UTR and one or more lncRNAs. Intermolecular base-pairing in humans involves Alu elements, which are a type of short interspersed repetitive element (SINE), whereas intermolecular base-pairing in rodents involves B and identifier SINEs. The STAU1 paralog STAU2, and STAU1 and STAU2 homodimerization and heterodimerization also promote SMD. In addition to mRNA−lncRNA interactions, they have found that mRNAs crosstalk in a way that involves direct mRNA−mRNA interactions between 3'UTR Alu elements in each mRNA, uncovering a new role for mammalian-cell mRNAs. This unexpected function, together with them discovering how STAU1 binding to inverted repeated 3'UTR Alu elements (IRAlus) (i) competes with nuclear (i.e., paraspeckle) retention mediated by p54nrb binding to 3'UTR IRAlus and also (ii) the repression of cytoplasmic translation mediated by PKR binding to 3'UTR IRAlus add new layers of complexity to the network of post-transcriptional interactions that regulate gene expression and involve ncRNA. They are in the process of purifying individual Alu element-containing lncRNAs together with the associated transcripts (both mRNAs and other lncRNAs) and proteins to gain insight into lncRNA structure and function.

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09:25-09:50 A bi-functional long non-coding RNA links neuronal differentiation to survival after stress

Dr Saba Valadkhan, Case Western Reserve University, USA

Abstract

To gain insight into the complex cellular roles performed by long non-coding RNAs, Dr Valadkhan analyzed the function of an intergenic lncRNA, BORG, which is almost exclusively expressed in neuronal cells in both mouse and human. Study of the function of BORG indicated that it is required for neuronal differentiation in mouse cell culture-based systems. In addition, they show that BORG plays a critical role in the stress response that was distinct from its neuronal differentiation function. Knock down and overexpression studies indicated a strong, positive correlation between the expression level of BORG and post-stress survival in cultured mammalian cells. In order to determine if BORG plays a role similar to what we have observed in cultured cells in vivo, they developed a transgenic mouse model that overexpresses BORG from a tet-regulated promoter. Induction of expression of BORG from the transgene in postnatal mice resulted in potentiation of the stress response, similar to what we had observed in cell culture-based systems. Despite inducing an increase in pro-survival factors, overexpression of BORG in vivo does not seem to be tumorigenic. Their observations indicate that both in vivo and in cultured cells, BORG RNA is a strong regulator of the stress response and suggest that the pro-survival function of BORG can have a protective effect against neurodegeneration and other conditions characterized by stress-mediated cell death.

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10:40-11:05 Structural architecture of lncRNAs in plants and mammals

Dr Karissa Sanbonmatsu, Los Alamos National Laboratory, USA

Abstract

Many genome-wide studies of long non-coding RNAs (lncRNAs) have been performed; however, few structural studies of individual lncRNAs exist. A clear consensus has not been achieved on the level of structure occurring in lncRNA systems. Structures are necessary not only to establish a mechanistic framework for lncRNAs, but also to establish the level of conservation of each lncRNA, since this cannot be achieved from sequence alone. Historically, secondary structures of the 16S and 23S rRNA provided an important framework for biochemical investigations and for structural studies of the ribosome, which is a 5-10 kB non-coding RNA, depending on species. Dr Sanbonmatsu establishes similar structural frameworks by determining secondary structures of individual lncRNA systems. In the case of smaller RNA systems, chemical probing has proved an instrumental methodology for directly determining secondary structures. In the case of larger systems, chemical probing alone has significant limitations due to the exponentially large number of possible secondary folds. To address this challenge, they developed Shotgun Secondary Structure (3S), a divide-and-conquer experimental strategy to derive secondary structures of lncRNAs. The 3S experimental strategy allows us to produce the secondary fold with minimal need for computational predictions. Here, they chemically probe the entire lncRNA transcript. Next, they repeat the probing on several overlapping fragments on the transcript. If the SHAPE profile for the fragment matches the profile for the same region of RNA in the context of the full transcript, this strongly suggests the fragment folds into a modular sub-domain or modular secondary motif, eliminating a large number of other possible folds. They applied this method to the Coolair lncRNA in A. thaliana and Braveheart in mouse, both of which possess clear phenotypes. They also applied the method to the human steroid receptor RNA activator (SRA-1). Coolair, Braveheart and SRA-1 each show highly structured sub-domains and share a unique expansive internal loop (‘right hand turn’ motif). They then use the secondary structure to find examples of the same lncRNA in other species, demonstrating conservation of structure.

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11:05-11:30 Long non-coding RNAs in cancer

Dr Sven Diederichs, University of Heidelberg, Germany

Abstract

The majority of the human genome is transcribed into non-protein-coding RNA. Hence, RNA is also the primary product of the cancer genome. Dr Diederichs lab have defined the ncRNA expression landscape of lung, breast and liver cancer providing a comprehensive expression map of over 17000 long ncRNAs and discovering new lncRNAs associated with cancer whose molecular and cellular functions they are currently elucidating. The nuclear lncRNA MALAT1 was one of the first lncRNAs associated with cancer: it is associated with metastasis development in lung cancer. However, its high abundance and nuclear localization have hampered its functional analysis. To uncover its functional importance, they developed a MALAT1 knockout model in human lung tumor cells by genomically integrating RNA destabilizing elements site-specifically into the MALAT1 locus. This approach yielded a 1000-fold silencing of MALAT1 providing a unique loss-of-function model. Proposed mechanisms of MALAT1 action include regulation of splicing or gene expression. In lung cancer, MALAT1 does not alter alternative splicing but regulates gene expression inducing a signature of metastasis-associated genes. Consequently, MALAT1-deficient cells are impaired in migration and form fewer tumor nodules in a mouse xenograft model. Encouraged by this discovery of the essential function of MALAT1 in lung cancer metastasis, they analyzed whether MALAT1 could serve as therapeutic target for an Antisense oligonucleotide (ASO). Notably, MALAT1-ASO treatment prevented metastasis formation after tumor implantation. Thus, targeting MALAT1 with ASOs provided a therapeutic approach to prevent lung cancer metastasis with MALAT1 serving as both, predictive marker and therapeutic target. In summary, ten years after the discovery of the lncRNA MALAT1 as a biomarker for lung cancer metastasis, their loss-of-function model unraveled the active function of MALAT1 as a regulator of gene expression governing hallmarks of lung cancer metastasis.

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11:50-12:30 Poster session

12:30-13:30 Lunch

13:30-17:00

Session 4: Non-conserved lncRNA roles in species uniqueness and disease

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Chairs

Professor Nicholas Proudfoot FRS, University of Oxford, UK

13:30-13:55 What hallmarks for long non-coding RNA? The mitochondrial connection

Dr Alexandra Henrion-Caude, Inserm, France

Abstract

A crucial role of mitochondria is to provide the universal currency for energy. This task forces them to be in tight communication with the rest of the cell machinery, and more specifically with the nucleus, which represents the control center. Connections take place not only through the cytoskeleton but also through intense genomic cross-talk with the nucleus, which provides mitochondria with nuclear-encoded proteins as well as non-coding RNAs that are required for mitochondrial homeostasis and function. Conversely, both strands of the mitochondrial DNA are entirely transcribed that generate also some non-coding sequences. Although information about mitochondrial long non-coding RNAs (lncRNAs) per se is still limited, candidates non-coding RNAs of various sizes recovered in mitochondrial fraction open up a rich field of information and investigation. Some insights as to mitochondrial lncRNAs as regulators of gene expression and as biomarkers will be presented. Some of these biological insights will hopefully find application in the steadily increasing number of pathologies that are related to dysfunctions of mitochondria, in conditions where pathogenicity and phenotypic expression are not readily understandable.

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13:55-14:20 LncRNAs and adaptation to environmental stress via post-transcriptional gene regulation

Dr James B. "Ben" Brown, Lawrence Berkeley National Laboratory, USA

Abstract

In phyla ranging from arthropods to mammals, lncRNAs are expressed in spatially restricted patterns – in one or a few tissues on average. Temporal expression patterns are similarly restricted; in Drosophila, expression during more than two hours of development is predominantly limited to neural tissues and the germ line. Environmental challenges reveal a host of largely non-coding genes that are not otherwise at detectable levels in any tissue or time point. In a diverse set of environmental challenges surveyed in Drosophila and Daphnia, lncRNAs are among the strongest and earliest responsive genes. Daphnia is an ecologically critical clade and forms the basis of the metazoan food web in over 90% of fresh water bodies on earth. Daphnia magna responds to microcystin (toxic algae; blooms cost the US >$2B per year) with the over-expression of a single gene, in which the longest ORF is 57aa and is not conserved. The modENCODE Consortium and the Consortium for Environmental Omics and Toxicology have generated large datasets interrogating the patterns of expression and interaction of lncRNAs. Dr Brown will discuss the developing picture of lncRNAs as rapidly evolving, adaptive molecules that help to shape the emergence of new ecological niches, as well as cell types and tissues, and hence, species.

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15:10-15:35 LncRNA regulators of metabolism

Professor Chris Ponting, University of Oxford

Abstract

Efficient enzymatic activity of the mitochondrial electron transport chain depends on replenishment of its subunits whose availability is determined by their rates of transcription, translation, import, and protein and mRNA turnover. We have shown that Cerox1, an unusually abundant and conserved long noncoding RNA (lncRNA), substantially alters complex I enzymatic activity, reactive oxygen species production, and levels of transcripts encoding 12 complex I subunits. These enzymatic changes are similar to those observed in disorders with mitochondrial dysfunction, such as Parkinson’s and Alzheimer’s diseases. Increased Cerox1 levels reduce cell division, protect cells against the cytotoxic effects of the complex I inhibitor rotenone and decoy microRNAs which otherwise reduce the abundance of complex I proteins. MicroRNA-488 overexpression reduces Cerox1 levels 13-fold, and mutation of its single response element in Cerox1 alters complex I transcripts’ levels. Cerox1 is the first lncRNA known to regulate mitochondrial energy metabolism.

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15:35-16:00 Primate-specific lncRNAs: from species phenotypic uniqueness to human disease

Professor Leonard Lipovich, Wayne State University, USA

Abstract

Long non-coding RNA (lncRNA) genes are at least as abundant as protein-coding genes in humans. LncRNA genes have low interspecies conservation: over 60% are not conserved beyond primates. Diverse regulatory roles – positive and negative, epigenetic and post-transcriptional, nuclear and cytoplasmic – have been identified for over a hundred human lncRNA genes to date, still a small number in contrast to the approximately 20,000 lncRNA genes discovered in the first one and a half decades of post-genomic biology. Our integration of the Gencode lncRNA resource with the NHGRI catalog of significant disease-associated SNPs from Genome-Wide Association Studies (GWAS) revealed specific diseases where a highly statistically significant portion of GWAS hits occurs in lncRNA exons, including obesity and several cancers. Notably, one obesity-associated lncRNA, LOC1572723, lacking conservation beyond primates, was independently pinpointed by 19 published GWAS datasets. Professor Lipovich pursued functional lncRNAomics in another lncRNA-associated disease – breast cancer – using the human estrogen receptor α positive cell line MCF7. Overexpression and knockdown of 26 estrogen-responsive lncRNAs shifted breast cancer cells along the apoptosis-proliferation axis. Reduced cell growth and increased cell death were consistently observed upon knockdown of estrogen-induced, and overexpression of estrogen-repressed, lncRNAs. 12 of these 26 lncRNAs originated after the prosimian split. Overexpression of BC041455, a primate-specific estrogen-repressed lncRNA, reduced ERK phosphorylation uniquely in ERα-positive cells, indicating modulation of the conserved MAP kinase pathway. Professor Lipovich shows by ribosome profiling that BC041455's polysome occupancy is distinct from that of highly translated mRNAs, with implications for hormone dependence of ectopic lncRNA translation in disease.

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16:20-17:00 Panel discussion

Long non-coding RNAs: evolution of new epigenetic and post-transcriptional functions

28-29 September 2015

Kavli Royal Society Centre, Chicheley Hall Newport Pagnell Buckinghamshire MK16 9JJ