Most “Dark Matter” Transcripts Are Associated With Known Genes

Short-read RNA sequencing in mouse and human tissues shows that most transcripts are encoded within or nearby known genes and that most of the genome is not transcribed.

Harm van Bakel1, Corey Nislow1,2, Benjamin J. Blencowe1,2, Timothy R. Hughes1,2*

1 Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada, 2 Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada


A series of reports over the last few years have indicated that a much larger portion of the mammalian genome is transcribed than can be accounted for by currently annotated genes, but the quantity and nature of these additional transcripts remains unclear. Here, we have used data from single- and paired-end RNA-Seq and tiling arrays to assess the quantity and composition of transcripts in PolyA+ RNA from human and mouse tissues. Relative to tiling arrays, RNA-Seq identifies many fewer transcribed regions (“seqfrags”) outside known exons and ncRNAs. Most nonexonic seqfrags are in introns, raising the possibility that they are fragments of pre-mRNAs. The chromosomal locations of the majority of intergenic seqfrags in RNA-Seq data are near known genes, consistent with alternative cleavage and polyadenylation site usage, promoter- and terminator-associated transcripts, or new alternative exons; indeed, reads that bridge splice sites identified 4,544 new exons, affecting 3,554 genes. Most of the remaining seqfrags correspond to either single reads that display characteristics of random sampling from a low-level background or several thousand small transcripts (median length = 111 bp) present at higher levels, which also tend to display sequence conservation and originate from regions with open chromatin. We conclude that, while there are bona fide new intergenic transcripts, their number and abundance is generally low in comparison to known exons, and the genome is not as pervasively transcribed as previously reported.

Author Summary 

The human genome was sequenced a decade ago, but its exact gene composition remains a subject of debate. The number of protein-coding genes is much lower than initially expected, and the number of distinct transcripts is much larger than the number of protein-coding genes. Moreover, the proportion of the genome that is transcribed in any given cell type remains an open question: results from “tiling” microarray analyses suggest that transcription is pervasive and that most of the genome is transcribed, whereas new deep sequencing-based methods suggest that most transcripts originate from known genes. We have addressed this discrepancy by comparing samples from the same tissues using both technologies. Our analyses indicate that RNA sequencing appears more reliable for transcripts with low expression levels, that most transcripts correspond to known genes or are near known genes, and that many transcripts may represent new exons or aberrant products of the transcription process. We also identify several thousand small transcripts that map outside known genes; their sequences are often conserved and are often encoded in regions of open chromatin. We propose that most of these transcripts may be by-products of the activity of enhancers, which associate with promoters as part of their role as long-range gene regulatory sites. Overall, however, we find that most of the genome is not appreciably transcribed.

Citation: van Bakel H, Nislow C, Blencowe BJ, Hughes TR (2010) Most “Dark Matter” Transcripts Are Associated With Known Genes. PLoS Biol 8(5): e1000371. doi:10.1371/journal.pbio.1000371

Academic Editor: Sean R. Eddy, HHMI Janelia Farm, United States of America

Received: December 3, 2009; Accepted: April 9, 2010; Published: May 18, 2010


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Changes in properties of wheat leaf cuticle during interactions with Hessian fly

The Plant Journal

Early View (Articles online in advance of print)

Published Online: 16 Apr 2010

Dylan K. Kosma 1,†,‡ , Jill A. Nemacheck 2,† , Matthew A. Jenks 1 and Christie E. Williams 2,3,*

  1 Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA ,   2 USDA-ARS Crop Production and Pest Control Research Unit, MWA, West Lafayette, IN 47907, USA , and   3 Department of Entomology, Purdue University, West Lafayette, IN 47907, USA
  *For correspondence (fax 765 494 5105; e-mail

  These authors contributed equally to this paper.

  Present address: Department of Plant Biology, Michigan State University, East Lansing, MI 48824, USA.


Infestation of wheat by Hessian fly larvae causes a variety of physical and biochemical modifications of the host plant. Changes occur in cuticle permeability, lipid composition and gene transcript abundance, and these responses differ substantially between resistant and susceptible wheat lines. Staining assays revealed that susceptible plants exhibited a generalized increase in leaf sheath epidermal permeability during infestation; whereas, epidermal permeability was only minimally affected in resistant plants. Furthermore, temporal profiling using gas chromatographic methods revealed that changes in cuticle lipid (wax and cutin) composition correlated well with differing levels of epidermal permeability in susceptible and resistant plants. Temporal analysis of cuticle-associated gene mRNA levels, by quantitative real-time PCR, indicated a relationship between transcript abundance and changes in cuticle lipid profiles of resistant and susceptible plants. Results suggest that conserving cuticle integrity via induction of specific wax constituents and maintenance of cutin amounts, determined by the accumulation of cuticle-associated transcripts, could be important components of wheat resistance to Hessian fly larvae.


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Posted by: shrikantmantri | May 18, 2010

GC3 biology in corn, rice, sorghum and other grasses

GC3 biology in corn, rice, sorghum and other grasses

Tatiana V Tatarinova email, Nickolai N Alexandrov email, John B Bouck email and Kenneth A Feldmann email

BMC Genomics 2010, 11:308doi:10.1186/1471-2164-11-308

Published: 16 May 2010

Abstract (provisional)


The third, or wobble, position in a codon provides a high degree of possible degeneracy and is an elegant fault-tolerance mechanism. Nucleotide biases between organisms at the wobble position have been documented and correlated with the abundances of the complementary tRNAs. We and others have noticed a bias for cytosine and guanine at the third position in a subset of transcripts within a single organism. The bias is present in some plant species and warm-blooded vertebrates but not in all plants, or in invertebrates or cold-blooded vertebrates.


Here we demonstrate that in certain organisms the amount of GC at the wobble position (GC3) can be used to distinguish two classes of genes. We highlight the following features of genes with high GC3 content: they (1) provide more targets for methylation, (2) exhibit more variable expression, (3) more frequently possess upstream TATA boxes, (4) are predominant in certain classes of genes (e.g., stress responsive genes) and (5) have a GC3 content that increases from 5' to 3'. These observations led us to formulate a hypothesis to explain GC3 bimodality in grasses.


Our findings suggest that high levels of GC3 typify a class of genes whose expression is regulated through DNA methylation or are a legacy of accelerated evolution through gene conversion. We discuss the three most probable explanations for GC3 bimodality: biased gene conversion, transcriptional and translational advantage and gene methylation.

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Science. 2010 May 14;328(5980):899-903.

Carbon dioxide enrichment inhibits nitrate assimilation in wheat and Arabidopsis.

Bloom AJ, Burger M, Rubio Asensio JS, Cousins AB.

Department of Plant Sciences, University of California at Davis, Davis, CA 95616, USA.


The concentration of carbon dioxide in Earth's atmosphere may double by the end of the 21st century. The response of higher plants to a carbon dioxide doubling often includes a decline in their nitrogen status, but the reasons for this decline have been uncertain. We used five independent methods with wheat and Arabidopsis to show that atmospheric carbon dioxide enrichment inhibited the assimilation of nitrate into organic nitrogen compounds. This inhibition may be largely responsible for carbon dioxide acclimation, the decrease in photosynthesis and growth of plants conducting C(3) carbon fixation after long exposures (days to years) to carbon dioxide enrichment. These results suggest that the relative availability of soil ammonium and nitrate to most plants will become increasingly important in determining their productivity as well as their quality as food.

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Science 30 April 2010:
Vol. 328. no. 5978, pp. 624 – 627
DOI: 10.1126/science.1187113


Lateral Transfer of Genes from Fungi Underlies Carotenoid Production in Aphids

Nancy A. Moran1,* and Tyler Jarvik2

Carotenoids are colored compounds produced by plants, fungi, and microorganisms and are required in the diet of most animals for oxidation control or light detection. Pea aphids display a red-green color polymorphism, which influences their susceptibility to natural enemies, and the carotenoid torulene occurs only in red individuals. Unexpectedly, we found that the aphid genome itself encodes multiple enzymes for carotenoid biosynthesis. Phylogenetic analyses show that these aphid genes are derived from fungal genes, which have been integrated into the genome and duplicated. Red individuals have a 30-kilobase region, encoding a single carotenoid desaturase that is absent from green individuals. A mutation causing an amino acid replacement in this desaturase results in loss of torulene and of red body color. Thus, aphids are animals that make their own carotenoids.

1 Department of Ecology and Evolutionary Biology, 1041 East Lowell Street, University of Arizona, Tucson, AZ 85721, USA.
2 Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, USA.

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GREAT improves functional interpretation of cis-regulatory regions

Journal name:Nature Biotechnology

Published online
02 May 2010

Cory Y McLean,Dave Bristor,Michael Hiller,Shoa L Clarke,Bruce T Schaar,Craig B Lowe,Aaron M Wenger& Gill Bejerano

We developed the Genomic Regions Enrichment of Annotations Tool (GREAT) to analyze the functional significance of cis-regulatory regions identified by localized measurements of DNA binding events across an entire genome. Whereas previous methods took into account only binding proximal to genes, GREAT is able to properly incorporate distal binding sites and control for false positives using a binomial test over the input genomic regions. GREAT incorporates annotations from 20 ontologies and is available as a web application. Applying GREAT to data sets from chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-seq) of multiple transcription-associated factors, including SRF, NRSF, GABP, Stat3 and p300 in different developmental contexts, we recover many functions of these factors that are missed by existing gene-based tools, and we generate testable hypotheses. The utility of GREAT is not limited to ChIP-seq, as it could also be applied to open chromatin, localized epigenomic markers and similar functional data sets, as well as comparative genomics sets.

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Plant Cell Physiol. 2010 Apr 7. [Epub ahead of print]

Two Closely Related Subclass II SnRK2 Protein Kinases Cooperatively Regulate Drought-Inducible Gene Expression.

Mizoguchi MUmezawa TNakashima KKidokoro STakasaki HFujita YYamaguchi-Shinozaki KShinozaki K.

RIKEN Plant Science Center, 3-1-1, Kouyadai, Tsukuba, Ibaraki 305-0074, Japan.


The subclass III group of SNF1-related protein kinase 2 (SnRK2) members is known to play an important role in ABA and osmotic stress signaling in Arabidopsis, however, roles of other subclasses are remained to be elusive. Here, we established a double mutant of SRK2C/SnR2.8 and SRK2F/SnRK2.7 to investigate functions of subclass II SnRK2s. Microarray analysis suggested that subclass II SnRK2s regulate some of drought-responsive genes involving AREB/ABF-type transcription factors and their targets, and quantitative RT-PCR confirmed those genes were down-regulated significantly in srk2cf. This study indicates that subclass II SnRK2s also play important roles in drought stress signaling in Arabidopsis.

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Nature 464, 721-727 (1 April 2010) | 


; Received 22 September 2008; Accepted 22 January 2010

Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes

Beate Neumann1,12, Thomas Walter1,12, Jean-Karim Hériché5,13, Jutta Bulkescher1, Holger Erfle1,3,13, Christian Conrad1,3, Phill Rogers1,13, Ina Poser6, Michael Held1,13, Urban Liebel1,13, Cihan Cetin3, Frank Sieckmann8, Gregoire Pau9, Rolf Kabbe10, Annelie Wünsche2, Venkata Satagopam4, Michael H. A. Schmitz7, Catherine Chapuis3, Daniel W. Gerlich7, Reinhard Schneider4, Roland Eils10, Wolfgang Huber9, Jan-Michael Peters11, Anthony A. Hyman6, Richard Durbin5, Rainer Pepperkok3 & Jan Ellenberg2

  1. MitoCheck Project Group,
  2. Gene Expression and,
  3. Cell Biology/Biophysics Units, Structural and,
  4. Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, D-69117 Heidelberg, Germany
  5. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1HH, UK
  6. Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany
  7. Institute of Biochemistry, Swiss Federal Institute of Technology Zurich (ETHZ), Schafmattstrasse 18, CH-8093 Zurich, Switzerland
  8. Leica Microsystems CMS GmbH, Am Friedensplatz 3, D-68165 Mannheim, Germany
  9. European Bioinformatics Institute, European Molecular Biology Laboratory, Cambridge CB10 1SD, UK
  10. Division of Theoretical Bioinformatics, German Cancer Research Center, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany
  11. Institute for Molecular Pathology, Dr Bohr Gasse 7, A-1030 Vienna, Austria
  12. These authors contributed equally to this work.
  13. Present addresses: MitoCheck Project Group, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, D-69117 Heidelberg, Germany (J.-K.H.); BIOQUANT Centre University Heidelberg, INF 267, D-69120 Heidelberg, Germany (H.E.); 3-V Biosciences GmbH, Wagistrasse 27, 8952 Schlieren, Switzerland (P.R.); Institute of Biochemistry, Swiss Federal Institute of Technology Zurich (ETHZ), Schafmattstrasse 18, CH-8093 Zurich, Switzerland (M.H.); Karlsruhe Institute of Technology KIT, Herrmann-von-Helmholtz Platz 1, D-76344 Eggenstein-Leopoldshafen, Germany (U.L.).

Correspondence to: Jan Ellenberg2 Correspondence and requests for materials should be addressed to J.E. (Email:


Despite our rapidly growing knowledge about the human genome, we do not know all of the genes required for some of the most basic functions of life. To start to fill this gap we developed a high-throughput phenotypic screening platform combining potent gene silencing by RNA interference, time-lapse microscopy and computational image processing. We carried out a genome-wide phenotypic profiling of each of the ~21,000 human protein-coding genes by two-day live imaging of fluorescently labelled chromosomes. Phenotypes were scored quantitatively by computational image processing, which allowed us to identify hundreds of human genes involved in diverse biological functions including cell division, migration and survival. As part of the Mitocheck consortium, this study provides an in-depth analysis of cell division phenotypes and makes the entire high-content data set available as a resource to the community.


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New defenses deployed against plant diseases

An international team led by scientists at the Sainsbury Laboratory in Norwich,UK, have transferred broad spectrum resistance against some important plant diseases across different plant families. This breakthrough provides a new way to produce crops with sustainable resistance to economically important diseases.

Food insecurity is driving the search for ways to increase the amount of food we grow, whilst at the same time reducing unsustainable agricultural inputs. One way to do this is to increase the innate ability of crops to fight off disease-causing pathogens. Increased disease resistance would reduce yield losses as well as reduce the need for pesticide spraying.

Breeding programs for resistance generally rely on single resistance genes that recognise molecules specific to particular strain of pathogens. Hence this kind of resistance rarely confers broad-spectrum resistance and is often rapidly overcome by the pathogen evolving to avoid recognition by the plant.

However, plants have another defence system, based on pattern recognition receptors (PRRs). PRRs recognise molecules that are essential for pathogen survival. These molecules are less likely to mutate without harming the pathogen's survival, making resistance to them more durable in the field. These essential molecules are common to many different microbes, meaning that if a plant recognises and can defend itself against one of these molecular patterns, it is likely to be resistant against a broad range of other pathogens.

Very few of these PRRs have been identified to date. Dr Cyril Zipfel and his group at the Sainsbury Laboratory in Norwich, UK, took a Brassica-specific PRR that recognises bacteria, and transformed it into the Solanaceae plants Nicotania benthaminia and tomato.

"We hypothesised that adding new recognition receptors to the host arsenal could lead to enhanced resistance," said Dr Zipfel.

Under controlled laboratory conditions, they tested these transformed plants against a variety of different plant pathogens, and found drastically enhanced resistance against many different bacteria, including some of great importance to modern agriculture such as Rastonia solanaceraum, the causal agent of bacterial wilt and a select agent in the United States under the Agricultural Bioterrorism Protection Act of 2002.

"The strength of this resistance is because it has come from a different plant family, which the pathogen has not had any chance to adapt to. Through genetic modification, we can now transfer this resistance across plant species boundaries in a way traditional breeding cannot," said Dr Zipfel.

Published in the journal Nature Biotechnology, the finding, that plant recognition receptors can be successfully transferred from one plant family to another provides a new biotechnological solution to engineering disease resistance. The Zipfel group is currently extending this work to other crops including potato, apple, cassava and banana that all suffer from important bacterial diseases, particularly in the developing world.

"A guiding principle in plant pathology is that most plants tend to be resistant to most pathogens. Cyril's work indicates that transfer of genes that contribute to this basic innate immunity from one plant to another can enhance pathogen resistance," commented Professor Sophien Kamoun, Head of the Sainsbury Laboratory. "The implications for engineering crop plants with enhanced resistance to infectious diseases are very promising."

This research was funded by the Gatsby Charitable Foundation and the Two Blades Foundation, who have patented the technology on behalf of the inventors, and involved research groups from INRA/CNRS in France, the University of California, Berkeley and Wageningen University in the Netherlands.


TSL Press Office: 
Andrew Chapple, Tel: 01603 251490, email: 
Zoe Dunford, Tel: 01603 255111, email:

Notes for Editors:

Reference: Inter-family transfer of a plant pattern recognition receptor confers broad-spectrum bacterial resistance, will be published online byNature Biotechnology on 14th March 2010. doi: 10.1038/nbt.1613

About The Sainsbury Laboratory

The Sainsbury Laboratory (TSL) is a world-leading research centre focusing on making fundamental discoveries about plants and how they interact with microbes. TSL is evolving its scientific mission so that it not only provides fundamental biological insights into plant-pathogen interactions, but also delivers novel, genomics-based, solutions which will significantly reduce losses from major diseases of food crops, especially in developing countries.

About The Two Blades Foundation

The Two Blades Foundation (2Blades) supports the development of durable disease resistances in crop plants and their deployment in agriculture. 2Blades is a US-based charitable organization that supports programs of research and development on durable disease resistance. Where research identifies ways of breeding for lasting resistance, 2Blades seeks to promote their deployment in practical programs of crop improvement. 2Blades aims to support the use of safe, environmentally-benign and sustainable strategies for crop production so as to provide long-term protection from crop losses due to plant disease. The Foundation recognises that there is an especially urgent need for the development of disease resistant crops in less developed and subsistence agricultures and consequently this is a major focus of its activity.

About the Gatsby Charitable Foundation:


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For two decades, scientists have been pursuing a potential new way to treat bacterial infections, using naturally occurring proteins known as antimicrobial peptides (AMPs). Now, MIT scientists have recorded the first microscopic images showing the deadly effects of AMPs, most of which kill by poking holes in bacterial cell membranes.

read more

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