Article

A Keystone for ncRNA

Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA 02142, USA. .
Genome biology (Impact Factor: 10.81). 05/2012; 13(5):315. DOI: 10.1186/gb-2012-13-5-315
Source: PubMed

ABSTRACT

A report on the Keystone symposium 'Non-coding RNAs' held at Snowbird, Utah, USA, 31 March to 5 April 2012.

Full-text

Available from: Ezgi Hacisuleyman, Mar 23, 2014
Once upon a time RNA fi t cleanly into the central dogma
as a messenger between DNA and protein. Over the past
50 years, RNA molecules have continually emerged as
dynamic and versatile regulators of the genome. Our
modern understanding of non-coding RNAs (ncRNAs)
may look like an intertwined mess of molecules, but
collectively they exhibit architecture and coordination,
leading to elegantly choreographed regulation of DNA
and protein by RNA.  e emerging role of RNA as an
orchestrator resonated throughout the inaugural Keystone
symposium on ncRNAs, with many examples of long
ncRNAs (lncRNAs) having critical roles across numerous
biological pathways. After the meeting, it was clear that
more surprises would emerge from the ‘dark matter’ of
the genome.
e keynote address by Nick Proudfoot (University of
Oxford) set the stage by describing how the most
classically studied regions of the genome, such as the β-
globin locus, are now emerging as being exquisitely
regulated by RNA. Specifi cally, Proudfoot summarized
years of work showing how the act of transcription
through non-coding regions, and importantly where
trans criptional termination occurs, regulates the epi-
genetic dynamics of the locus. Intriguingly, convergent
transcription by RNA polymerase II (RNA pol II) may
serve as a substrate to recruit Dicer and other factors of
the RNA interference (RNAi) machinery. Similarly,
Robert Martienssen (Cold Spring Harbor Laboratory)
presented an interplay between RNA/DNA polymerase
activity and RNAi in establishing heterochromatic
domains.  e dependence on co-transcriptional RNAi
allows the release of RNA polymerase and prevents
collision with the centromeric DNA replication machinery.
Together these studies demonstrate the need for not only
identifying lncRNAs involved in epigenetic establishment
but also for understanding many simultaneous inter-
twined layers of regulation.
The human noncoding transcriptome reveals a
map of ‘noncodarnia
omas Gingeras (Cold Spring Harbor Laboratory)
provided an overview of the complexity of the human
transcriptome resulting from the eff orts of the ENCODE
consortium.  e transcriptomic map has gained an
unprecedented resolution, revealing that 76% of our
genome is transcribed. With an average of approximately
eight transcripts per genic region, the wealth of ENCODE
has redefi ned the ‘one gene - one function’ hypothesis
into ‘many transcripts - one function, or possibly many.
Using complementary datasets and approaches, Piero
Carninci and the Riken OMICs Center have provided
new insights into lncRNA promoter regulation. By fi ne
mapping of the 5’ 7-methyl guanosine caps on RNA, the
group have found that 6 to 30% of 5’ start sites of mouse
and human transcripts initiate within repetitive elements.
Remarkably, over 250,000 retrotransposon-derived trans-
cription start sites show tissue- and cell-compartment-
specifi c expression.
Leonard Lipovich (Wayne State University) and
colleagues added 6,000 lncRNAs to this catalog by
examining unclassifi ed human cDNA clones and their
expression profi les to determine whether these lncRNAs
contribute to neurological disease phenotypes.  ey
found that certain primate-specifi c and non-conserved
lncRNAs are diff erentially expressed in brain regions that
show high levels of activity. Some of these lncRNAs,
antisense to protein-coding genes, can regulate their
neighbors’ expression. Weaving the intricacy of the trans-
criptome with the complexity of the mammalian body
development and cognition, John Mattick (Garvan
Institute of Medical Research) presented examples that
emphasized the need to further understand the diversity
of lncRNAs. Digging into the depths of the ‘dark matter
in the genome’ using capture enrichment methods
revealed not only numerous novel lncRNAs and their
Abstract
A report on the Keystone symposium ‘Non-coding
RNAs held at Snowbird, Utah, USA, 31 March to 5 April
2012.
Keywords Non-coding RNA, epigenetics, chromatin
© 2010 BioMed Central Ltd
A Keystone for ncRNA
Ezgi Hacisuleyman
1,2,3
, Moran N Cabili
2,3,4
and John L Rinn
2,3
*
MEETING REPORT
*Correspondence: john_rinn@harvard.edu
3
Department of Stem Cell and Regenerative Biology, Harvard University,
Cambridge, MA 02138, USA
Full list of author information is available at the end of the article
Hacisuleyman et al. Genome Biology 2012, 13:315
http://genomebiology.com/2012/13/5/315
© 2012 BioMed Central Ltd
Page 1
isoforms but also isoforms of well-studied protein-coding
mRNAs such as p53. Hundreds of lncRNAs were shown
to change during stem cell diff erentiation and to have
similar transcript stability to mRNAs, and many are
associated with epigenetic complexes, suggesting that
this complexity cannot be dismissed en masse as trans-
criptional noise.
RNA-RNA interactions
An ever emerging theme is the importance of RNA-RNA
interactions and gene regulation. Kevin Morris ( e
Scripps Research Institute) described new fi ndings on
lncRNA-directed epigenetic regulation through RNA-
RNA interactions. Morris and colleagues observed an
antisense transcript from the PTEN pseudogene
(PTENpg1 asRNA), which is transcribed in the opposite
direction to the previously reported PTENpg1 sense
transcript (which can sequester microRNAs and aff ects
PTEN translation rates).  e PTENpg1 asRNA seems to
direct transcriptional gene silencing of PTEN by
interacting with the DNA methyltransferase Dnmt3a and
the histone-lysine N-methyltransferase Ezh2 and aff ect-
ing their localization to the PTEN promoter. Moreover,
the PTENpg1 asRNA (containing a poly(A) tail) seems to
facilitate the cellular localization of PTENpg1 sense
transcript (lacking a poly(A) tail).  us, this pseudogene
node seems to control PTEN at both the translational
and the transcriptional level.
ncRNAs in cellular and developmental biology
David Spector (Cold Spring Harbor Laboratory) and
Shinichi Nakagawa (RIKEN Advanced Science Institute)
presented a comprehensive investigation of the physio-
logical roles of lncRNAs in mouse model systems. Using
knockout mice, Spector and colleagues concluded that
the lncRNA Malat1 was physiologically dispensable
under laboratory conditions.  is lack of phenotype may
be attributed to genetic redundancy or stress conditions
that were not examined. Indeed, Nakagawa examined the
physiological role of the nuclear lncRNA Neat1 (which is
essential for the formation of nuclear structures involved
in splicing termed paraspeckles) using knockout mice
and observed a phenotype only in adult homozygote
females, which became infertile at an earlier age. Seth
Blackshaw (John Hopkins University School of Medicine)
presented the Six3OS RNA as another example of a
lncRNA required in developmental processes. Loss and
gain of function of Six3OS, which is transcribed in the
oppo site direction from the Six3 transcription factor
(TF) mRNA, showed that Six3OS regulates retinal cell
specifi cation. Several elegant follow-up experiments
suggest that Six3OS regulates Six3-dependent
transcription by recruiting the Polycomb repressive
complex 2 (PRC2) to Six3 target genes.
Martin Walsh (Mount Sinai School of Medicine)
presented his recent work on the complex interplay
between RNA pol II and pol III, whereby RNA pol III can
aff ect transcription by accentuating the function of RNA
pol II. Ablation of the components of RNA pol III caused
a signifi cant depletion of an enhancer ncRNA in the Sox2
locus followed by changes in chromatin signatures.
Further analysis of the three-dimensional spacing of RNA
pol III sites suggest that RNA pol III-regulated trans-
cription could have a role in chromatin architecture,
especially in the context of embryonic stem cell
diff erentiation.
Rachel Du é (Institut Curie) demonstrated how
lncRNAs could control diff erential methylation patterns
and imprinting during germ layer specifi cation. Duffi é
presented a biallelically expressed lncRNA that was suffi -
cient to induce DNA methylation in cis. Carla Klattenhoff
(Massachusetts Institute of Technology) presented
braveheart, a lncRNA required for stem cell diff eren-
tiation into cardiomyocytes. Loss of braveheart causes
disorganization and lack of beating in myofi brils and
shorter sarcomeres. Collectively, these studies demon-
strate the importance of lncRNAs in establishing cellular
identity.
RNA and chromatin
e intersection of RNA with chromatin was introduced
by the well-studied example of Xist by Neil Brockdorff
(University of Oxford) and Jeannie Lee (Massachusetts
General Hospital). Brockdorff showed that Xist RNA
associates directly with additional proteins such as
heterogeneous nuclear ribonucleoprotein U (hnRNPU)
and PRC1. Brockdorff and colleagues have also identifi ed
a distinct form of PRC1, in which RING1 and YY1
binding protein (Rybp) replaces the catalytic Cbx subunit
and associates with Xist RNA, suggesting that in addition
to PRC2, hnRNPU and PRC1 are also involved in
localizing Xist RNA to the inactive X. Lee further
elucidated the mechanism by which the Xist lncRNA
controls the silencing on the inactive X. Allele-specifi c
localization profi les of PRC2 during X inactivation suggest
a highly selective localization: fi rst, PRC2 localizes to
limited number of nucleation spots and, as Xist RNAs
spreads in cis, PRC2 is recruited to additional sites in a
gradient, unlike recruitment on autosomes.
As an attempt to generalize the observation that
ncRNAs such as Xist bind the PRC2 and recruit it to its
target loci, Richard Jenner (University College London)
and colleagues observed short RNAs that are transcribed
from CpG islands surrounding PRC2-bound promoters.
Jenner’s group showed that these short RNAs are
required for effi cient methylation of histone H3 on lysine
27 (H3K27me3) through interaction with a newly charac-
terized amino-terminal domain of Suppressor of zeste 12
Hacisuleyman et al. Genome Biology 2012, 13:315
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(Suz12). Collectively, these studies clearly demonstrate
the interplay between DNA, lncRNAs, and proteins to
establish proper epigenetic states and cellular physiology.
With the goal of identifying where RNA localizes on
chromatin, Matthew Simon (Massachusetts General
Hospital) and Jason West (Massachusetts General Hospital)
introduced a new method (CHART) to purify regions
directly bound by lncRNAs. CHART successfully
identifi ed the protein and chromatin targets of the para-
speckle-associated lncRNA NEAT1 in human cells. Initial
analysis of CHART coupled with sequencing revealed the
chromosomal binding sites of NEAT1 and suggested that
NEAT1 facilitates tertiary DNA inter actions involved in
nuclear architecture.
Deepening our understanding of recruitment of
chromatin modifi ers by lncRNAs, Howard Chang (Stan-
ford University) and colleagues discovered the lncRNA
HOTTIP, which activates the expression of its neigh-
boring genes in cis by recruiting the activating modifi er
WDR5. Using crystallography and site-specifi c muta-
genesis, the group identifi ed the RNA-binding domain of
WDR5. Moreover, binding to HOTTIP regulates the
stability of WDR5, suggesting a model in which a RNA-
based molecular clock regulates protein complex
formation and stability. Similar to HOTTIP, Ramin
Shiekhattar (Wistar Institute) presented a set of lncRNAs
with enhancer-like functions. Shiekhattar described a
specifi c example, in which an lncRNA enhances the
expression of genes in cis through physical interactions
with the Mediator complex.  e lncRNA-Mediator
interaction facilitates DNA looping to activate distal
genes. Together, these suggest that lncRNAs emanat-
ing from enhancer regions may have RNA-based
functions.
Using a detailed map of large intergenic ncRNAs
(lincRNAs), one of us (JR) and colleagues used a
computational ‘guilt-by-association approach’ to identify
lincRNAs involved in specifi c pathways. One such
example led to the discovery of a lncRNA, Required for
adipogenesis 1 (RAP1). RAP1 contains a striking feature:
multiple repetitive exonic sequences that physically
associate with hnRNPU. Single-molecule RNA localiza-
tion methods showed that RAP1 shows nuclear locali-
zation indicative of large chromosomal domains. Using a
similar guilt-by-association approach, Keith Vance (Uni-
ver sity of Oxford) from Chris Ponting’s group used
computational and evolutionary genomics to identify
mouse lincRNAs close to genes encoding developmental
TFs. One such lincRNA is Plinc, which can act in cis to
repress its bidirectionally promoted neighbor Pax6, as
well as in trans to repress other targets involved in cell
cycle and synaptic function. Plinc is enriched on
chromatin and associates with the transcriptional
cofactors Kap1 and Erh.
Non-mammalian ncRNA
ncRNAs also have critical regulatory roles in prokaryotes.
Gisela Storz (National Institute of Child Health and
Human Development) gave several examples from
bacteria, in which small RNAs act in parallel to TFs to
ne-tune the TFs’ target gene levels through a base-
pairing mechanism. In parallel, Jennifer Doudna
(University of California, Berkeley) gave new insights into
the CRISPR adaptive immune system in bacteria, in
which viral DNA segments are integrated within bacterial
DNA repeat clusters and are later transcribed to short
RNAs that act in a viral defense pathway. Doudna
highlighted some similarities between eukaryotic RNAi
and prokaryotic CRISPR target recognition mechanisms
based on molecular structures of these machineries.
RNA and disease
Andrew Feinberg (John Hopkins University School of
Medicine) presented a comprehensive analysis of the
epigenetic changes in cancer. Surveying diff erential DNA
methylation in cancer revealed the loss of DNA methy la-
tion of the lncRNA termed HOTS (H19 opposite tumor
suppressor) encoded antisense to the maternally im-
printed H19 gene, which encodes an untranslated RNA
of unknown function. Feinberg described large lamin-
associated DNA domains that are also lost in cancer.
Similar to HOTS, almost all genes have naturally
occurring antisense transcripts (NATs) that are ripe for
RNA-RNA interactions and are often misregulated in
disease states. Claes Wahlestedt (University of Miami)
and colleagues have developed single-stranded oligo-
nucleotides termed antagonists of NATs (antagoNATs)
that upregulate specifi c endogenous genes in vitro and in
vivo by interfering with the function of NATs. As NATs
are widely expressed, the approach holds promise for
potential RNA-based therapeutics. Together these
studies highlight the need to understand the link between
epigenetic regulation and lncRNAs in the etiology of
disease.
Trans-generational RNA for the future
Paramutation is a form of non-Mendelian heredity in
which the phenotypic expression of an allele is mutated
while its genotype is intact.  e meeting concluded with
exciting updates on the current understanding of this
phenomenon from worms to mammals. Andrew Spence
(University of Toronto) described how a maternal trans-
cript of the sex-determining gene fem-1 does not require
any coding capacity to trigger fem-1 expression, which it
does by preventing RNA-dependent epigenetic silencing
of this gene. Oded Rechavi (Columbia University Medical
Center) demonstrated how virus-derived small inter fer-
ing RNAs can be trans-generationally transmitted in a
non-Mendelian manner. Finally, Minoo Rassoulzadegan
Hacisuleyman et al. Genome Biology 2012, 13:315
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Page 3
(University of Nice) showed evidence for RNA-induced
hereditary transmission of variation aff ecting the
phenotype of three diff erent genes in mouse animals. By
electroporating an inducer small RNA homologous to the
gene of interest to embryonic stem cells, Rassoulzadegan
and colleagues were able to obtain a paramutable eff ect
over many passages in cell culture. Collectively, these
studies have provided exciting new evidence for the
potential role of RNA in trans-generational inheritance
in mammals.
is meeting was a resounding success and clearly
demonstrated the versatility, diversity, and importance of
ncRNAs in gene regulatory programs. John Mattick
ended the meeting with inspiring words, highlighting the
imminence of many groundbreaking discoveries to come
from the dark matter of the genome. Kevin Morris
reinforced the need for a collaborative spirit among the
ncRNA community, which will rapidly drive the fi eld to a
greater success. One thing was clear: the transcriptome
has revealed many mysterious creatures that comprise
the mysterious world of ‘Noncodarnia.
Author details
1
Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138,
USA.
2
Broad Institute of Massachusetts Institute of Technology and Harvard,
Cambridge, MA 02142, USA.
3
Department of Stem Cell and Regenerative
Biology, Harvard University, Cambridge, MA 02138, USA.
4
Department of
Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
Published: 25 May 2012
doi:10.1186/gb-2012-13-5-315
Cite this article as: Hacisuleyman E, et al.: A Keystone for ncRNA. Genome
Biology 2012, 13:315.
Hacisuleyman et al. Genome Biology 2012, 13:315
http://genomebiology.com/2012/13/5/315
Page 4 of 4
Page 4
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    [Show abstract] [Hide abstract] ABSTRACT: Abstract Objective: To identify differentially expressed long non-coding RNA genes in human myometrium in women in spontaneous labor at term. Materials and Methods: Myometrium was obtained from women undergoing cesarean deliveries who were not in labor (n=19) and women in spontaneous labor at term (n=20). RNA was extracted and profiled using an Illumina(®) microarray platform. We have used computational approaches to bound the extent of long non-coding RNA representation on this platform, and to identify co-differentially expressed and correlated pairs of long non-coding RNA genes and protein-coding genes sharing the same genomic loci. Results: We identified co-differential expression and correlation at two genomic loci that contain coding-lncRNA gene pairs: SOCS2-AK054607 and LMCD1-NR_024065 in women in spontaneous labor at term. This co-differential expression and correlation was validated by qRT-PCR, an independent experimental method. Intriguingly, one of the two lncRNA genes differentially expressed in term labor had a key genomic structure element, a splice site that lacked evolutionary conservation beyond primates. Conclusions: We provide for the first time evidence for coordinated differential expression and correlation of cis-encoded antisense lncRNAs and protein-coding genes with known, as well as novel roles in pregnancy in the myometrium of women in spontaneous labor at term.
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