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Title: V6 RNA world


1
V6 RNA world
short name full name function mRNA, rRNA, tRNA,
you know them well ... snRNA small nuclear
RNA splicing and other functions snoRNA small
nucleolar RNA nucleotide modification of
RNAs Long ncRNA Long noncoding
RNA various miRNA MicroRNA gene
regulation siRNA small interfering RNA gene
regulation

SS 2009 lecture 6
Biological Sequence Analysis
1
2
RNA structure
Single stranded RNA molecules frequently require
a specific tertiary structure. The scaffold for
this structure is provided by secondary
structural elements which are H-bonds within the
molecule. This leads to several recognizable
"domains" of secondary structure like hairpin
loops, bulges and internal loops. RNA hairpin
2RLU Stem loop 1NZ1

www.rcsb.org
SS 2009 lecture 6
Biological Sequence Analysis
2
3
miRNAs
microRNAs (miRNA) are single-stranded RNA
molecules of 21-23 nucleotides in length, which
regulate gene expression. miRNAs are encoded by
DNA but not translated into protein (non-coding
RNA). Instead, each primary transcript (a
pri-miRNA) is processed into a short stem-loop
structure called a pre-miRNA and finally into a
functional miRNA. Mature miRNA molecules are
partially complementary to one or more messenger
RNA (mRNA) molecules. Their main function is to
down-regulate gene expression of this mRNA.

www.wikipedia.org
SS 2009 lecture 6
Biological Sequence Analysis
3
4
snRNAs
  • Small nuclear RNA (snRNA) are found within the
    nucleus of eukaryotic cells.
  • They are transcribed by RNA polymerase II or RNA
    polymerase III and are involved in a variety of
    important processes such as
  • RNA splicing,
  • regulation of transcription factors or RNA
    polymerase II, and
  • maintaining the telomeres.
  • They are always associated with specific
    proteins. The complexes are referred to as small
    nuclear ribonucleoproteins (snRNP) or sometimes
    as snurps.
  • A large group of snRNAs are known as small
    nucleolar RNAs (snoRNAs).
  • These are small RNA molecules that play an
    essential role in RNA biogenesis and guide
    chemical modifications of rRNAs, tRNAs and
    snRNAs.
  • They are located in the nucleolus and the cajal
    bodies of eukaryotic cells.

www.wikipedia.org
SS 2009 lecture 6
Biological Sequence Analysis
4
5
Long ncRNAs
Long noncoding RNAs (long ncRNAs) are non-protein
coding transcripts longer than 200 nucleotides.
This limit is due to practical considerations
including the separation of RNAs in common
experimental protocols. Additionally, this
limit distinguishes long ncRNAs from small
regulatory RNAs such as miRNAs, short interfering
RNAs (siRNAs), Piwi-interacting RNAs (piRNAs),
snoRNAs etc.

www.wikipedia.org
SS 2009 lecture 6
Biological Sequence Analysis
5
6
XIST
One prominent well-understood ncRNA is the ncRNA
XIST that silences the inactive X chromosome by
the ncRNA XIST. To normalize the copy number of
X chromosomes between male and female cells,
transcription of XIST RNA from one of the two
female X chromosome is involved in recruiting
Polycomb group proteins (PcG) to trimethylate
histone H3 on lysine 27 (H3K27me3), rendering the
chromosome transcriptionally silent. (wait for
second half of this lecture). PcGs thereby
remodel chromatin. Polycomb Repressive Complex 2
(PRC2) is a PcG that is comprised of H3K27
histone methyl transferase (HMTase) EZH2 and core
components Suz12 and EED, apparently initiates
this histone modi?cation. Subsequently, Polycomb
Repressive Complex 1 (PRC1) is another PcG that
maintains this modi?cation and promotes chromatin
compaction.

Plath, Science 300, 131 (2003)
SS 2009 lecture 6
Biological Sequence Analysis
6
7
Hotair another important ncRNA
Because many HMTase complexes lack DNA-binding
domains but possess RNA-binding motifs, it has
been postulated that ncRNAs may guide speci?c
histone modi?cation activities to discrete
chromatin loci. Rinn et al. showed that HOTAIR
ncRNA binds PRC2 and is required for robust H3K27
trimethylation and transcriptional silencing of
the HOXD locus. Hox genes are a group of
related genes that specify the anterior-posterior
axis and segment identity of metazoan organisms
during early embryonic development. These genes
are critical for the proper number and placement
of embryonic segment structures (such as legs,
antennae, and eyes). HOTAIR may therefore be one
of the long-sought-after RNAs that interface the
Polycomb complex with target chromatin.

Rinn, Cell 129, 1311(2007)
SS 2009 lecture 6
Biological Sequence Analysis
7
8
Conservation of Hotair
HOTAIR is expressed in posterior and distal
sites, indicating the conservation of anatomic
expression pattern from development to
adulthood. Interestingly, this transcript has
very high nucleotide conservation in vertebrates
(99.5, 95, 90, and 85 sequence identity in
chimp, macaque, mouse, and dog genomes,
respectively). Yet it contains many stop codons
with little amino acid sequence conservation
amongst vertebrates. These results suggest that
HOTAIR may function as a long ncRNA.

Rinn, Cell 129, 1311(2007)
SS 2009 lecture 6
Biological Sequence Analysis
8
9
Hotair mechanism
A potentially attractive model of epigenetic
control is the programming of active or silencing
histone modi?cations by speci?c noncoding RNAs.
Just as transcription of certain ncRNA can
facilitate H3K4 methylation and activate
transcription of the downstream Hox genes,
distant transcription of other ncRNAs may target
the H3K27 HMTase PRC2 to speci?c genomic sites,
leading to silencing of transcription and
establishment of facultative heterochromatin.

Model of long ncRNA regulation of chromatin
domains via histone-modi?cation enzymes.
Transcription of ncRNAs in cis may increase the
accessibility of TrxG proteins such as the
histone methyl transferase ASH1 or MLL or
directly recruit them, leading to H3K4
methylation and transcriptional activation of the
downstream HOX gene(s). In contrast, recruitment
of PRC2 is programmed by ncRNAs produced in
trans, which targets PRC2 activity by
as-yet-incompletely-de?ned mechanisms to target
loci. PRC2 recruitment leads to H3K27 methylation
and transcriptional silencing of neighboring HOX
genes.
Rinn, Cell 129, 1311(2007)
SS 2009 lecture 6
Biological Sequence Analysis
9
10
What is epigenetics?
Epigenetics refers to alternate phenotypic states
that are not based in differences in genotype,
and are potentially reversible, but are generally
stably maintained during cell division. Examples
imprinting, twins, cancer vs. normal cells,
differentiation, ... The narrow interpretation
of this concept is that of stable differential
states of gene expression. A much more expanded
view of epigenetics has recently emerged in which
multiple mechanisms interact to collectively
establish - alternate states of chromatin
structure, - histone modification, - associated
protein composition, - transcriptional activity,
and - in mammals, cytosine-5 DNA methylation at
CpG dinucleotides.

Laird, Hum Mol Gen 14, R65 (2005)
11
Basic principles of epigeneticsDNA methylation
and histone modfications
The human genome contains 23 000 genes that must
be expressed in specific cells at precise times.
Cells manage gene expression by wrapping DNA
around clusters (octamers) of globular histone
proteins to form nucleosomes. These nucleosomes
of DNA and histones are organized into chromatin,
the building block of a chromosome.

Rodenhiser, Mann, CMAJ 174, 341 (2006)
Bock, Lengauer, Bioinformatics 24, 1 (2008)
12
Epigenetic modifications

Strands of DNA are wrapped around histone
octamers, forming nucleosomes. These nucleosomes
are organized into chromatin, the building block
of a chromosome. Reversible and site-specific
histone modifications occur at multiple sites
through acetylation, methylation and
phosphorylation. DNA methylation occurs at
5-position of cytosine residues within CpG pairs
in a reaction catalyzed by DNA methyltransferases
(DNMTs). Together, these modifications provide a
unique epigenetic signature that regulates
chromatin organization and gene expression.
Rodenhiser, Mann, CMAJ 174, 341 (2006)
13
Cytosine methylation
3-6 of all cytosines are methylated in human
DNA. How many cytosines are in normal
DNA? How many CpG islands are in normal DNA?

Mammalian genomes contain much less (only 20-25
) of the CpG dinucleotide than is expected by
the GC content. This is typically explained in
the following way As most CpGs serve as targets
of DNA methyltransferases, they are usually
methylated. 5-Methylcytosine, whose occurrence
is almost completely restricted to CpG
dinucleotides, can easily deaminate to thymine.
If this mutation is not repaired, the affected
CpG is permanently converted to TpG (or CpA if
the transition occurs on the reverse DNA strand).
Hence, methylCpGs represent mutational hot
spots in the genome. If such mutations occur in
the germ line, they become heritable. A
constant loss of CpGs over thousands of
generations can explain the scarcity of this
special dinucleotide in the genomes of human and
mouse.
Esteller, Nat. Rev. Gen. 8, 286 (2007)
14
effects in chromatin organization affect gene
expression

Schematic of the reversible changes in chromatin
organization that influence gene expression
genes are expressed (switched on) when the
chromatin is open (active), and they are
inactivated (switched off) when the chromatin is
condensed (silent). White circles unmethylated
cytosines red circles methylated cytosines.
Rodenhiser, Mann, CMAJ 174, 341 (2006)
15
Basic principles of epigeneticsDNA methylation
and histone modfications
Changes to the structure of chromatin influence
gene expression genes are inactivated (switched
off) when the chromatin is condensed (silent),
and they are expressed (switched on) when
chromatin is open (active). These dynamic
chromatin states are controlled by reversible
epigenetic patterns of DNA methylation and
histone modifications. Interestingly,
repetitive genomic sequences are heavily
methylated, which means transcriptionally
silenced. Enzymes involved in this process
include - DNA methyltransferases (DNMTs), -
histone deacetylases (HDACs), - histone
acetylases, - histone methyltransferases and the
- methyl-binding domain protein MECP2.

Rodenhiser, Mann, CMAJ 174, 341 (2006)
16
DNA methylation
Typically, unmethylated clusters of CpG pairs are
located in tissue-specific genes and in essential
housekeeping genes, which are involved in routine
maintenance roles and are expressed in most
tissues. These clusters, or CpG islands, are
targets for proteins that bind to unmethylated
CpGs and initiate gene transcription. In
contrast, methylated CpGs are generally
associated with silent DNA, can block
methylation-sensitive proteins and can be easily
mutated. The loss of normal DNA methylation
patterns is the best understood epigenetic cause
of disease. In animal experiments, the removal
of genes that encode DNMTs is lethal in humans,
overexpression of these enzymes has been linked
to a variety of cancers.

Rodenhiser, Mann, CMAJ 174, 341 (2006)
17
Uptake of methyl groups
DNA methylation patterns fluctuate in response to
changes in diet, inherited genetic polymorphisms
and exposures to environmental chemicals. Methyl
groups are acquired through the diet and are
donated to DNA through the folate and methionine
pathways. Consequently, changes in DNA
methylation may occur as a result of low dietary
levels of folate, methionine or selenium. This
can lead to diseases such as neural tube defects,
cancer and atherosclerosis. Imbalances in
dietary nutrients can lead to hypomethylation
(which contributes to improper gene expression)
and genetic instability (chromosome
rearrangements). E.g. hyperhomocysteinemia and
global hypomethylation have been observed in
vitro in atherosclerosis models. In advanced
stages of atherosclerosis, hyperproliferation may
further contribute to DNA hypomethylation and
altered gene expression.

Rodenhiser, Mann, CMAJ 174, 341 (2006)
18

Esteller, Nat. Rev. Gen. 8, 286 (2007)
19

Esteller, Nat. Rev. Gen. 8, 286 (2007)
20
Epigenetic regulation during development

Surani, Hayashi, Hajkova, Cell 128, 747 (2007)
21
Epigenetic regulation during development

Surani, Hayashi, Hajkova, Cell 128, 747 (2007)
22
Epigenetic regulation during development

Surani, Hayashi, Hajkova, Cell 128, 747 (2007)
23
Epigenetic signals in ES cells and in
differentiated cells

Bernstein, Meissner, Lander, Cell 128, 669 (2007)
24
Large scale identification of functional ncRNA

Eric Lander, Whitehead Institute, MIT
John Rinn, Harvard Medical School
Gringeras, Nat. Biotech. 27, 346
(2009) Commentary on Guttman article
SS 2009 lecture 6
Biological Sequence Analysis
24
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