Table Of ContentAhmad M. Khalil Editor
Molecular
Biology of Long
Non-coding
RNAs
Second Edition
Molecular Biology of Long Non-coding RNAs
Ahmad M. Khalil
Editor
Molecular Biology of Long
Non-coding RNAs
Second Edition
Editor
Ahmad M. Khalil
Case Western Reserve University School of Medicine
Cleveland, OH, USA
ISBN 978-3-030-17085-1 ISBN 978-3-030-17086-8 (eBook)
https://doi.org/10.1007/978-3-030-17086-8
© Springer Nature Switzerland AG 2019
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Preface
Since the dawn of molecular biology it was accepted that most, if not all, biological
processes are controlled by proteins. With a few exceptions, the vast majority of the
scientific literature from the 1960s to the 2000s focused on proteins and their roles
in development and disease. Although as early as the 1960s it was clear that living
cells produce RNA molecules that are never translated into proteins (e.g., ribosomal
RNAs, transfer RNAs), the prevailing dogma during that time was that RNA only
serves as messengers for protein translation (mRNAs) or as constituents of protein
building machinery (rRNAs, tRNAs). In the late 1980s and early 1990s, with the
discovery of H19 and XIST, two genes that code for long non-coding RNAs with no
protein-coding capacity, it was the first emerging evidence that RNAs could serve
regulatory roles.
Over three decades after the discoveries of H19 and XIST, we now know that the
human genome is transcribed into both coding and non-coding transcripts, with cur-
rent estimates of over 20,000 long non-coding RNAs. These coding and non-coding
transcripts represent the first level of functional biology downstream of DNA and
that, both directly and indirectly, yield the astounding complexity of molecular biol-
ogy within a cell. While historically the bulk of scientific research focused on the
coding portion of the transcriptome, only 2% of our DNA encodes such protein-
synthesizing transcripts. Our understanding of the functional roles of non-coding
transcripts has evolved slowly, first with the roles of ribosomal and transfer RNAs
in protein synthesis, and more recently with the regulatory roles of microRNAs and
long non-coding RNAs (lncRNAs) on gene expression.
lncRNAs are transcripts greater than 200 nucleotides in length, which lack
protein- coding capacity. These RNA polymerase II transcripts are capped, spliced,
and poly-adenylated, yet many remain localized to the nucleus. With ~20,000
lncRNAs identified in the human transcriptome, many have now been shown to be
functional and have important biological roles; however, much more remains to be
discovered about the myriad of roles they play in human biology. Our focus here is
to explore a sampling of the stories that this rich field of research continues to pro-
duce and to give our readers a broad sense of the importance of lncRNAs to human
biology.
v
vi Preface
At the time of this book publication, we can identify lncRNA functional roles in
transcriptional regulation, subnuclear body formation and function, signaling mod-
ulation, RNA decoy action, and as protein scaffolds or protein interaction modula-
tors with outcomes impacting epigenetic regulation, stem cell maintenance, DNA
damage response regulation, developmental specification, X-chromosome inactiva-
tion (XCI), and many other cellular functions. With only a fraction of lncRNAs
characterized to date, the list of functional roles for lncRNAs will continue to
expand alongside our understanding of their biological importance.
One of the most exemplary stories of lncRNA biology is that of XCI. XCI is the
mammalian mechanism for achieving dosage compensation of sex-linked genes and
results in the complete transcriptional silencing of most genes on a single X chro-
mosome in XX females. Several lncRNAs play central roles in XCI, most impor-
tantly the X inactive specific transcript (XIST), which coats the inactive X
chromosome and silences transcription. This 17 kb transcript has multiple isoforms,
some of which are poly-adenylated, but maintains strict nuclear localization, con-
tains six functional domains, and lacks conserved open reading frames. XIST pro-
vides a prime example of a lncRNA-modulating gene expression in healthy tissues
and is required for normal development.
lncRNAs differ somewhat from proteins on an evolutionary scale, as they dem-
onstrate markedly less primary sequence conservation and greater tissue specificity.
Together with their critical roles across much of cellular biology, it is tempting to
speculate that some of the diversity of eukaryotic life might have emerged via the
evolution of lncRNAs. Cancer biology offers insight into this concept, as lncRNAs
are common players in oncogenic gene expression dysregulation. Examples of
lncRNAs in critical roles promoting the hallmarks of cancer include the lncRNA
LUNAR1 cis upregulation of IGF1-mediated growth signaling (sustained prolifera-
tive signaling) and aberrant expression of the lncRNA telomerase RNA component
(TERC), which provides the template for telomere extension (enabling replicative
immortality). Comparing cancer to adjacent normal tissues also provides opportu-
nity to elucidate lncRNA roles in healthy physiology, for instance, the growth
arrest-specific 5 (GAS5) is a lncRNA whose expression is downregulated in several
cancers. GAS5 binds to glucocorticoid receptor and stochastically inhibits interac-
tions with its ligands, thereby blocking downstream anti-apoptotic regulators. While
these examples provide insight into the diverse functions of lncRNAs, they also
provide examples of therapeutic targets and biomarkers that are expressed in a more
tissue- specific manner than proteins.
In the second edition of this book, we have gathered experts from across the
world to detail the involvement of lncRNAs in human cancers, XCI, cardiovascular
disease, nuclear organization, and the chemical modifications of RNAs. Through
these detailed chapters, we offer insights into our rapidly growing understanding of
the significance of lncRNAs to the whole of human biology and perhaps even inspire
new endeavors of study into this rapidly expanding field.
Cleveland, OH, USA Daniel Vail
Ahmad M. Khalil
Contents
Complex Regulation of X-Chromosome Inactivation
in Mammals by Long Non-coding RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
J. Mauro Calabrese
Chemical Modifications and Their Role in Long
Non-coding RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Sindy Zander, Roland Jacob, and Tony Gutschner
Long Non-coding RNAs and Nuclear Body Formation
and Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Alina Naveed, Ellen Fortini, Ruohan Li, and Archa H. Fox
New Insights into the Molecular Mechanisms of Long Non-coding
RNAs in Cancer Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Ligia I. Torsin, Mihnea P. Dragomir, and George A. Calin
The Role of Long Non-coding RNAs in Melanoma
Genesis and Progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Piyush Joshi and Ranjan J. Perera
Long Non-coding RNAs in the Development and Maintenance
of Lymphoid Malignancies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Melanie Winkle, Agnieszka Dzikiewicz-Krawczyk, Joost Kluiver,
and Anke van den Berg
Long Non-coding RNAs in Vascular Health and Disease . . . . . . . . . . . . . . 151
Viorel Simion, Stefan Haemmig, and Mark W. Feinberg
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
vii
Complex Regulation of X-Chromosome
Inactivation in Mammals by Long
Non-coding RNAs
J. Mauro Calabrese
1 Introduction
Female mammals silence the majority of genes along one of their two X chromo-
somes in a process termed X-chromosome inactivation (XCI). XCI likely evolved in
mammals as the X and Y chromosome, once homologous autosomal pairs, diverged
in sequence, largely through degeneration of the Y. This degeneration left males
with only one functional copy of most X-linked genes, necessitating the develop-
ment of a compensation process that would equalize X-linked gene dosage between
the sexes (Livernois et al. 2012).
XCI is critical for mammalian development. Severe defects in the process are
developmentally lethal, while abnormalities in X-chromosome dosage, which occur
in about 1 of 500 live births, can be pleiotropic disorders, associated with forms of
intellectual disabilities, infertility, and autoimmunity (Powell 2005). The impor-
tance of regulating X-linked gene dosage is underscored by the chromosomal count-
ing process inherent to XCI. Regardless of the total number of X chromosomes an
individual has, XCI ensures that one X per diploid genome remains active, with
the remainder subject to inactivation, in both males and females. For example,
XCI tends to silence two Xs in tetraploid female cells and only one in tetraploid
male/female cell fusions (Monkhorst et al. 2008). In both cases, the ratio of one
active X per diploid genome is maintained. Similarly, in humans, XCI shuts down
two Xs in females with three (triple X syndrome) and one X in males with two
(Klinefelter’s syndrome); the sole X in females with Turner’s syndrome remains
active. These chromosomal abnormalities are often accompanied by chronic health
issues (Powell 2005), indicating imperfect regulation of X-linked dosage. However,
the intrinsic capability of mammalian cells, male or female, to sense and at least
J. M. Calabrese (*)
Department of Pharmacology, Lineberger Comprehensive Cancer Center,
University of North Carolina, Chapel Hill, NC, USA
e-mail: [email protected]
© Springer Nature Switzerland AG 2019 1
A. M. Khalil (ed.), Molecular Biology of Long Non-coding RNAs,
https://doi.org/10.1007/978-3-030-17086-8_1
2 J. M. Calabrese
partially deal with abnormalities in X-chromosome dosage is remarkable and speaks
to the physiological importance of XCI.
In addition to its role in development and human health, XCI has long been a
paradigm for epigenetic silencing mediated by long non-coding RNAs (lncRNAs),
given the critical role of Xist and other lncRNAs in the process. Advances in DNA
sequencing technologies have led to the identification of thousands of lncRNAs
expressed by the mammalian genome, many of which are developmentally regu-
lated and conserved (Cabili et al. 2011; Derrien et al. 2012; Dunham et al. 2012;
Hezroni et al. 2015; Iyer et al. 2015). Early studies have shown these RNAs can
function in a range of biological processes, including stem cell maintenance, regula-
tion of the DNA damage response, and developmental specification (Kopp and
Mendell 2018; Ulitsky and Bartel 2013). XCI was one of the first identified gene
regulatory processes in mammals with a conserved role for lncRNAs (Brockdorff
et al. 1992; Brown et al. 1992). Therefore, as the importance of lncRNA-mediated
gene regulation has come into focus, XCI has remained a flagship model for under-
standing lncRNA function and mechanism of action. In the pages below, we describe
the major features of XCI, with particular focus on the roles that lncRNAs play in
the process.
2 XCI Overview
In the mouse, historically the field’s most utilized experimental model, XCI occurs
in two waves during early development. The first is termed imprinted XCI, due to
the exclusive inactivation of the paternally inherited X chromosome (Takagi and
Sasaki 1975). Imprinted XCI occurs rapidly after formation of the zygote, initiating
at the 4-cell stage of development and nearing completion for some paternal loci at
the formation of the early blastocyst, around the 32-cell stage (Kalantry et al. 2009;
Okamoto et al. 2005; Patrat et al. 2009; Williams et al. 2011). This stark parent-of-
origin bias appears to be independent of meiotic sex chromosome inactivation that
occurs in the male germ line (Okamoto et al. 2005) and instead is due to an imprint
placed on the maternal X during oocyte maturation, which somehow blocks XCI
from occurring on the chromosome (Tada et al. 2000). Cells of the extraembryonic
lineage propagate a paternally derived inactive X (Xi) throughout their existence
(Takagi and Sasaki 1975; West et al. 1977). In contrast, XCI is reversed in the inner
cell mass (ICM) of the blastocyst, which gives rise to the embryo proper (Mak et al.
2004; Okamoto et al. 2004). Post-implantation, XCI re-occurs in the epiblast, nearing
completion around embryonic gestational day (E) 6.5 (Rastan 1982). In this second
wave, termed random XCI, the choice to inactivate a given X is largely random and
independent from its parent-of-origin (McMahon et al. 1983). XCI is then maintained
in all cells save those from the germ line (Sugimoto and Abe 2007), resulting in adult
females who are mosaics of paternally and maternally derived Xis.
Not all mammals share the biphasic inactivation strategy of the mouse. While
rats and cows show imprinted XCI in their extraembryonic tissue (Wake et al. 1976;
Complex Regulation of X-Chromosome Inactivation in Mammals by Long Non-coding… 3
Xue et al. 2002), suggesting a mouselike biphasic inactivation strategy, other eutherian
mammals examined to date – humans, horses, and mules – appear to undergo ran-
dom XCI in all lineages (Moreira de Mello et al. 2010; Wang et al. 2012). In contrast,
metatherians, such as the kangaroo and opossum, inactivate their paternally inherited
X in all tissues (Grant et al. 2012; Sharman 1971).
3 Control of XCI via the X-Inactivation Center
Studies of balanced chromosomal translocations in the mouse mapped the location
of a single X-linked region that invariably tracked with inactivation of adjoining
X-linked DNA and often led to partial silencing of the fused autosome (Lyon et al.
1989). Because of the region’s ability to inactivate neighboring DNA, it was pro-
posed to contain the cis-mediated genetic signals required to initiate and maintain
XCI and was termed the X-inactivation center (Xic) (Fig. 1a; Rastan and Brown 1990).
Fig. 1 Xist and the X-inactivation center. (a) The protein coding genes, non-coding RNAs, and
regulatory elements of the murine X-inactivation center, depicted to scale relative to UCSC
genome build mm9. Genes and regulatory regions in black text denote those discussed in the text
with documented or proposed roles in XCI. Genes in gray text have no known roles in XCI. Exons
and introns are depicted as solid bars and hashed lines, respectively. Regulatory regions are
depicted as colored bars above genes. Denoted TADs are those described in Nora et al. (2012). The
large blue bar spanning the majority of (a) denotes the genomic span of bacterial and yeast artifi-
cial chromosomes that recapitulate aspects of XCI when integrated as multi-copy transgene arrays
into mouse cell lines (Heard et al. 1999; Lee et al. 1996). (b, c) Mouse and human Xist genomic
loci. Exons and introns are depicted as in (A). Exonic regions in gray mark the location of the six
annotated Xist repeats, A through F, as described in Brockdorff et al. (1992), Brown et al. (1992),
and Nesterova et al. (2001). The location of the RepA transcript within the murine Xist locus is
underlined
