lncRNAs in development and disease: from functions to mechanisms

Differential expression of long non-coding RNAs (lncRNAs) during differentiation and their misregulation in cancer highlight their potential as cell fate regulators. While some example lncRNAs have been characterized in great detail, the functional in vivo relevance of others has been called into question. Finding functional lncRNAs will most probably require a combination of complementary approaches that will greatly vary depending on their mode of action. In this review, we discuss the different tools available to dissect genetically lncRNA requirements and how each is best suited to studies in particular contexts. Moreover, we review different strategies used to select candidate lncRNAs and give an overview of lncRNAs described to regulate development and cancer through different mechanisms.M.J.D. was funded by a PhD fellowship from Boehringer Ingelheim Fonds, a graduate studies fellowship from ‘la Caixa’ Foundation and by the Watson School of Biological Sciences. G.J.H. is a Wellcome Trust Investigator, was an investigator of the Howard Hughes Medical Institute, and is supported by Cancer Research UK, The Royal Society (Wolfson Professorship) and a generous gift from Kathryn W. Davis

Genome and transcriptome of the regeneration-competent flatworm, Macrostomum lignano.

The free-living flatworm, Macrostomum lignano has an impressive regenerative capacity. Following injury, it can regenerate almost an entirely new organism because of the presence of an abundant somatic stem cell population, the neoblasts. This set of unique properties makes many flatworms attractive organisms for studying the evolution of pathways involved in tissue self-renewal, cell-fate specification, and regeneration. The use of these organisms as models, however, is hampered by the lack of a well-assembled and annotated genome sequences, fundamental to modern genetic and molecular studies. Here we report the genomic sequence of M. lignano and an accompanying characterization of its transcriptome. The genome structure of M. lignano is remarkably complex, with ∼75% of its sequence being comprised of simple repeats and transposon sequences. This has made high-quality assembly from Illumina reads alone impossible (N50=222 bp). We therefore generated 130× coverage by long sequencing reads from the Pacific Biosciences platform to create a substantially improved assembly with an N50 of 64 Kbp. We complemented the reference genome with an assembled and annotated transcriptome, and used both of these datasets in combination to probe gene-expression patterns during regeneration, examining pathways important to stem cell function.This work is supported by National Institutes of Health Grants R37 GM062534 (to G.J.H.) and R01-HG006677 (to M.S.); National Science Foundation Grant DBI-1350041 (to M.S.); and a Swiss National Science Foundation Grant 31003A-143732 (to L.S.). This work was performed with assistance from Cold Spring Harbor Laboratory Shared Resources, which are funded, in part, by Cancer Center Support Grant 5P30CA045508.This is the final version of the article. It first appeared from PNAS via http://dx.doi.org/10.1073/pnas.151671811

lncRNA Spehd Regulates Hematopoietic Stem and Progenitor Cells and Is Required for Multilineage Differentiation.

Long non-coding RNAs (lncRNAs) show patterns of tissue- and cell type-specific expression that are very similar to those of protein coding genes and consequently have the potential to control stem and progenitor cell fate decisions along a differentiation trajectory. To understand the roles that lncRNAs may play in hematopoiesis, we selected a subset of mouse lncRNAs with potentially relevant expression patterns and refined our candidate list using evidence of conserved expression in human blood lineages. For each candidate, we assessed its possible role in hematopoietic differentiation in vivo using competitive transplantation. Our studies identified two lncRNAs that were required for hematopoiesis. One of these, Spehd, showed defective multilineage differentiation, and its silencing yielded common myeloid progenitors that are deficient in their oxidative phosphorylation pathway. This effort not only suggests that lncRNAs can contribute to differentiation decisions during hematopoiesis but also provides a path toward the identification of functional lncRNAs in other differentiation hierarchies.This work was supported by Cancer Research UK. We specifically thank the Cancer Research UK Cambridge Institute Biological Resource Unit, Flow Cytometry, Research Instrumentation, Light Microscopy and Genomics Cores for their support throughout this project. M.J.D was supported by a PhD Fellowship from the Boehringer Ingelheim Fonds. G.J.H. is a Wellcome Trust Investigator, Royal Society Wolfson Research Professor, and was a Howard Hughes Medical Institute Investigator

Yeast cultures with UCP1 uncoupling activity as a heating device

7 páginas, 5 figuras, 3 tablas -- PAGS nros. 300-306Uncoupling proteins (UCPs) are mitochondrial transporters that facilitate controlled dissipation of the proton gradient and thus regulate energetic efficiency. The heat generating capacity of UCP from brown adipose tissue was investigated in yeasts expressing the protein recombinantly under conditions in which the temperature of the growth medium was measured directly. A Liquid Culture Calorimeter (LCC) was built consisting of a thermally isolated culture flask able to keep yeast cultures warm without resorting to additional heating. The exact internal temperature of the cultures was monitored for 24 h through a thermocouple connected to a data logger. Under these conditions, significant temperature increases (1 °C) in the media were recorded when yeast strains expressing endogenously active UCP1 mutants were grown. This is the first direct evidence, in a eukaryotic microbial model, of a temperature rise associated with uncoupling activity, and could be seen as the first step toward developing a biological heating deviceThis work was presented as ‘The Hot Yeast Project’ by the Valencia team in the 2008 iGEM competition. The team was supported by Vicerrectorado de Investigación, Desarrollo e Innovación (Universidad Politécnica de Valencia), Ingenio Mathematica i-math Project and European community's Seventh Framework Programme (FP7/2007-2013) under grant agreement no 212894 ‘Targeting environmental pollution with engineered microbial systems à la carte’ (TARPOL). Research was financially supported by Vicerrectorat d’Investigació (Universitat de València). Manuel Porcar has a ‘Ramon y Cajal’ research contract and Emilio Navarro a ‘Juan de la Cierva’ research contract both from the Spanish Ministerio de Ciencia e Innovación. Eduardo Rial is supported by research grants from the Ministerio de Ciencia e Innovación (BFU2006-08182 and Consolider-Ingenio 2010 CSD2007-00020Peer reviewe