Andrea Hand
11/4/11
BIOL 303
Term Paper
Discovering the Functions of LincRNA
In 2007, popular opinion was that lincRNA, large intergenic noncoding RNA, were
transcriptional noise. To determine if these long intergenic noncoding RNAs were functional,
mice lincRNA DNA from four different cell types was compared to human lincRNA DNA
(Ponjavic, et al., 2007). The hypothesis was that if lincRNA is functional, then the sequence of
base pairs in the regions from which the RNA is transcribed will be the same between mammal
species. Over time as the two species separated, lincRNA’s DNA sequence would have to remain
the same to maintain lincRNA’s function, and so the mutation rate would be lower in these
segments of DNA than in segments that are nonfunctional.
To determine if the DNA of the promoter and sequence was maintained, the amount of
substitutions, insertions and deletions, and the locations in which splicing took place were
compared. They compared the two species’ mutation rates in noncoding ancestral regions to the
mutation rates in the DNA that gave rise to these noncoding RNA, and found that the mutation
rates were significantly lower for the noncoding RNA DNA. For many of the noncoding RNA
coding regions, substitution and transversion rates were between 3% and 40% lower. For those
same coding regions, insertion and deletion rates, as well as splicing site movement rate were
also lower. These regions, which had a large amount of guanine and cytosine and therefore
would be expected to have an elevated mutation rate, had the same mutation rate as the areas
around them, showing that selection was going on to reduce mutation to retain functionality. This
research suggested that these previously purposeless RNA were meaningful and called for
further inquiry (Ponjavic, et al., 2007).
Actively transcribed regions have histone H3 lysine 4 trimethylation (K3) in the promoter
region, and a longer stretch of DNA with histone H3 lysine 36 trimethylation (K36) (Guttman et
al., 2009). Therefore, regions of transcribed DNA denoted by an K3-K36 stretch were compared
to the known protein coding regions, and those that did not likely code for proteins were
proposed to be lincRNA. Around 3,300 human lincRNA were found through this method from
only 6 human cell types (Khalil et al., 2009).
A lincRNA called HOTAIR had recently been found to bind to a polycomb repressive
complex, PRC2, and other chromatin modifying complexes (Rinn et al. 2007), and so Khalil et
al. (2009) immunoprecipitated multiple chromatin modifying complexes and determined that
38% of the lincRNAs were physically associated with at least one of the chromatin modifying
complexes. Short interference RNAs (siRNA) were used to deplete lincRNAs that were
associated with PRC2 to determine if their presence affected gene expression in relation to
PRC2. It was found that genes repressed by PRC2 were activated upon removal of lincRNA,
implying that lincRNAs have a function in gene regulation (Khalil et al., 2009), a far step from
the transcription noise they were supposed to be two years earlier.
A year later, Guttman et al., (2010) examined transcribed RNAs from three mouse cell
types for the presence of splice sites. Breaking the exons up, the DNA that the lincRNAs were
transcribed from could be found, which led to the identification of 1140 lincRNAs that had
previously been overlooked. They were overlooked before because the DNA was examined for
regions that were transcribed, instead of the reverse process. These lincRNAs were missed in
previous studies for multiple reasons. Some of these lincRNAs were closer to coding regions and
so excluded from the search in other studies, however it was determined that these stretches of
DNA were unlikely to be coding for proteins and so were added to the lincRNA list. Some were
previously overlooked because they were smaller than some other lincRNAs, although they were
all by definition greater than two hundred base pairs long, and many were expressed at lower
levels due to less chromatin enrichment- which led to the earlier oversight as they searched for
long stretches of K3-K36 histones (Guttman et al., 2010).
This study determined that the average lincRNA is made up of 3.7 exons, each exon an
average 350 base pairs, and an average mature spliced size of 1.3 kb. Proteins generally have 9.7
exons, with each exon 291 base pairs, and a mature length of 2.9 kb. LincRNA levels in cells are
only slightly lower than protein coding mRNA levels, and lincRNAs undergo alternative splicing
just as mRNAs do (Guttman et al., 2010). Even though lincRNAs do not code for proteins, they
have been conserved in many mammal species, are similar in many ways to mRNAs, and affect
gene expression in multiple known ways, and probably in many more ways that are currently
unknown.
One specific way that lincRNA functions is in the p-53 pathway that arrests cell change
or causes apoptosis when DNA is damaged, which is important for tumor suppression. Many
lincRNAs are affected by the p-53 pathway, this research focused upon lincRNA-p21, called that
because it is located near a protein called p-21. It was determined that p53 could bind directly to
lincRNA-p21’s promoter region by comparing the structure of the p-53 protein and the promoter
region of the RNA (Huarte, et al., 2010). When LincRNA-p21 was targeted by RNAi (interfering
RNA), gene expression levels in the p-53 pathway changed, proving it had an effect. Huarte, et
al. (2010) also found that heterogenous nuclear ribonucleoprotein K, hnRNP-K, binds to
lincRNA-p21, and so tests were done to determine their relationship. It was found that the 5’ end
of the lincRNA allowed hnRNP-K to bind to DNA segments and regulate gene expression and in
other undetermined ways works in the regulation of gene apoptosis (Huarte, et al., 2010).
This year, a paper on the effects of lincRNAs on embryonic stem cells was published
(Guttman, et al., 2011). The goal was to determine the validity of the hypothesis that lincRNAs
were transcription byproducts. The main method to determine if and how lincRNAs function was
through the insertion of short hairpin RNA complementary to lincRNA segments into ES cells to
knockdown the effectiveness of individual lincRNAs, which would cause genes they had
repressed to be overexpressed, and the opposite for those genes they had enhanced. Since the
loss of individual lincRNAs affected gene expression to a similar extent as protein loss, it was
apparent that lincRNAs were functional, and not byproducts as had been suggested. In addition,
the hypothesis that lincRNAs only affect nearby genes in cis, was also easily refuted, supporting
the hypothesis that lincRNAs act in trans.
To determine how lincRNAs affect the pluripotent state, shRNAs were added to cells
containing a luciferase reporter gene downstream of the Nanog protein promoter, Nanog a
protein marker of the pluripotent state. Knockdown of many lincRNAs led to changes in Nanog
levels to an extent similar to when Nanog and other protein pluripotency regulators were
knocked down. In addition, more than 90% of the lincRNA knockdowns led to the loss of
embryonic stem cell morphology. Knockdowns of different lincRNAs lead to the expression of
markers for different differentiation pathways. Markers indicating differentiation into endoderm,
ectoderm, neuroectoderm, mesoderm, and trophectoderm cell types were each observed upon
lincRNA knockdowns indicating that lincRNAs repressed differentiation from the pluripotent
state. In addition, Guttman et al. (2011) demonstrated that approximately 75% of the 226
lincRNA promoters tested were bound by at least 1 of 9 pluripotency transcription factors
indicating that transcription of these lincRNA genes is regulated by at least some of the same
molecules as the proteins known to maintain the pluripotent state.
The research mentioned here and much more shows that lincRNA have significant roles
in gene expression, and very few of the proposed 4,500 lincRNA have yet to be examined. In
these few cell types, it has been found that lincRNAs are integral in tumor suppression and
maintenance of the pluripotent state. In addition, they have been conserved in many mammal
species, are processed by splicing pathways, are present at levels similar to that of mRNA, and
interact with chromatin regulation proteins. Thus, although the roles of lincRNAs are not fully
understood, and neither is the mechanism in which they affect gene expression, great strides have
been made to elucidate the importance of these abundant RNAs.
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