SS 2009 – lecture 6 Biological Sequence Analysis 1 V6 RNA world short namefull namefunction mRNA,...

SS 2009 – lecture 6Biological Sequence Analysis

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


2

RNA structure

www.rcsb.org

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


3

miRNAs

www.wikipedia.org

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.


4

snRNAs

www.wikipedia.org

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.


5

Long ncRNAs

www.wikipedia.org

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.


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XIST

Plath, Science 300, 131 (2003)

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 modification.

Subsequently, Polycomb Repressive Complex 1 (PRC1) is another PcG that

maintains this modification and promotes chromatin compaction.


7

Hotair: another important ncRNA

Rinn, Cell 129, 1311(2007)

Because many HMTase complexes lack DNA-binding domains but possess

RNA-binding motifs, it has been postulated that ncRNAs may guide specific

histone modification 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.


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Conservation of Hotair

Rinn, Cell 129, 1311(2007)

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.


9

Hotair mechanism

Rinn, Cell 129, 1311(2007)

A potentially attractive model of epigenetic control is the programming of active

or silencing histone modifications by specific 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 specific genomic sites, leading

to silencing of transcription and establishment of facultative heterochromatin.

Model of long ncRNA regulation of chromatin domains via histone-modification 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-defined mechanisms to target loci. PRC2 recruitment leads to H3K27 methylation and transcriptional silencing of neighboring HOX genes.

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)

Basic principles of epigenetics:DNA 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)

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)

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?

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

Mammalian genomes contain much less (only 20-25 %) of the CpG dinucleotide than is expected by the

G+C 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.

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)

Basic principles of epigenetics:DNA 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)

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.


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.


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

Epigenetic regulation during development

Surani, Hayashi, Hajkova, Cell 128, 747 (2007)

Epigenetic signals in ES cells and in differentiated cells

Bernstein, Meissner, Lander, Cell 128, 669 (2007)


24

Large scale identification of functional ncRNA

Gringeras, Nat. Biotech. 27, 346 (2009)Commentary on Guttman article

John Rinn, Harvard Medical School

Eric Lander, Whitehead Institute, MIT

SS 2009 – lecture 6 Biological Sequence Analysis 1 V6 RNA world short namefull namefunction mRNA,...

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