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Non-coding RNAs

or RNAs come more than in three flavours...

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How big part of human transcribed

RNA results in proteins?

Of all RNA, transcribed in higher eukaryotes, 98%

are never translated into proteins

Of those 98%, about 50-70% are introns

The rest originate from non-protein genes,

including rRNA, tRNA and a vast number of other

non-coding RNAs (ncRNAs)

Even introns have been shown to contain ncRNAs,for example snoRNAs

It is thought that there might be order of 10,000

different ncRNAs in mammalian genome

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The two main classes of ncRNAs

Housekeeping ncRNAs, which are constitutively

expressed and required for normal function and

viability of cell Regulatory ncRNAs are expressed only in certain

stages of organism development or as a response

to external stimuli.

Regulatory ncRNAs can affect the expression of

other genes at the level of transcription or

translation

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Housekeeping ncRNAs

tRNA and rRNA - translation

snRNA Pre-mRNA splicing

snoRNA rRNA modification

gRNA guide RNA in RNA editing

Telomerase RNA

primer for telomericDNA synhesis

A few other...

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4.5S RNA and 7S RNA a part of signal recognition particle

(SRP)

SRP recognizes signalling amino acid sequence in the N-terminus of growingpolypeptide chain

Upon signal recognition, ribosome is attached to endoplasmatic reticlum so

that the protein, made by ribosome, enters the secretory pathway

ribosome

Signalsequence

Endoplasmatic reticlum membrane

mRNASRP

translocon SRP

SRPreceptor

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Structure of SRP

RNA holds together the protein subunits of

SRP as well as helps to bind to ribosome

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tmRNA and trans-translation

tmRNA is a hybrid molecule, half tRNA,

half mRNA

tmRNA helps to rescue ribosomes, bound to

mRNA which lacks the termination codon

In addition, tmRNA adds a degradation

signal to nascent protein

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3

If the termination codon by some mistake is not reached, the ribosome

gets stack upon the reaching of 3 end of mRNA and has to be rescued

Since the stop codon is not reached, the newly made protein is

probably wrong and needs to be degraded

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The tRNA part of tmRNA (black) adds an alanine to thegrowing polypeptide chain

The mRNA part (red) enters the ribosome and the

synthesis of polypeptide is continued with aid of normal

tRNAs (blue), until the termination codon is reached In the end, ribosome is released and the newly made fusion

protein is degraded due to the signal sequence in C-

terminus

3

Ala

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Regulatory ncRNAs

Transcriptional regulators

Translational regulators

Modulators of protein function

Regulators of RNA and protein distribution

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Dosage compensation

In animals, males and females have

different number of X chromosomes (e.g. 1

or 2)

To equalize the expression levels from X

chromosome in males and females some

sort of mechanism must exist, called dosagecompensation

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Dosage compensation mechanisms

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The role of roX ncRNAs in dosage

compensation inDrosophila

MSL-1

MSL-3

MOFhistone

acetylase

MLE

helicase

roX1/roX2

ncRNAs

MSL-2

roX1 and roX2 ncRNAs are

expressed only in males and they

are responsible for for MSL (Male

Specific Lethal) complex assembly.

The MSL complex acetylates H4histones on X chromosomes

therefore increasing the

transcription level

MSL complex has about 35 entry

sites in Drosophila genome. Two of

them actually contain roX1/rox2

gene. This suggests a possible role

of rox1/rox2 RNAs in entry site

recognition

2 x MSL-3

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Acetylated

lysines

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Silencing of one female X chromosome

in mammals

The X chromosome silencing is mediated by Xist a 16,000 ntlong ncRNA

Xist ncRNA recruited complex has one entry site in X

chromosome, corresponding to Xist gene itself Xist appears to recruit a specific histone isoform H2A1.2

which maintains the chromosome in inactive state

Additionally, Xist containing complexes recruit histone

deacetylases and methylases Xist activity is regulated by another 40,000 nt long ncRNA

Tsix, which contains anti-sense sequence of Xist and therefore isable to regulate Xist activity by base-pairing to it

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Genetic imprinting and shRNAs

Genetic imprinting is a process which results in expressionon only one allele of gene, while the allele originating fromthe other parent is silenced

Process is somewhat similar to dosage compensation

The differences of expression from both alleles are due todifferent states of chromatin (euchromatin andheterochromatin) and also to differential methylation ofDNA

Activity of small heterochromatic RNAs (shRNAs) appearto be essential for establishing and maintaining theimprinted status of genes

Activity of various shRNAs is not limited only to genetic

imprinting

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DNA and RNA recognition models of shRNA

initiated chromatin condensation

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Translational regulation

Translational regulation by ncRNAs is

achieved by anti-sense mechanism, when

ncRNA binds to target mRNA RNA interference covered separately in

the end of this lecture

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Translation of human HFE gene is downregulated by

anti-sense RNA

HFEpromoter

Anti-sensepromoter

Sense exons

Anti-senseexons

Ribosome

HFE mRNAA

Anti-sense RNA

B

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DsrA RNA inE.coli activates ribosome binding to

stress-response s factor rpoS mRNA

Ribosome binding site

blocked by base-pairing

rpoS mRNA

DsrA RNA

RBS accessible

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Protein function modulation

Some ncRNAs can bind directly to proteins,

altering their structure, enzymatic activities

or ligand binding Targets of such ncRNAs often are proteins,

involved in transcription, for example

nuclear receptors or general transcriptionfactors

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6S RNA modulates s70 function inE.coli

+ + +s70RNA pol

6S RNA

Log-phase Stationary phase

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Ribozymes

RNA molecules with catalytical properties (Ribonucleic acidenzymes)

In nature ribozymes occur mostly within self-splicing intrones andRNA encoded parasites satellites and viroids

The catalyzed reactions in naturally occuring ribozymes are limited

to cleavage and ligation of RNA Some researchers consider even ribosomes being ribozymes, since

the peptide bond formation is catalyzed by RNA

Most naturally occuring ribozymes act on themselves

The catalytical efficiency of ribozymes is typically much lower(~1000-fold) than of analogous protein enzymes

Several synthetic ribozymes are cabaple of performing otherreactions than RNA cleavage and ligation

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Cleaving ribozymes

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The general secondary structure of

hammerhead ribozyme

Y=C or T, R=A or G, H=A,T or C

Dot represents any nucleotide

Cleavage

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The 3D structure of hammerhead ribozyme

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Hammerhead ribozyme

mechanism

Requires bivalent metal ion for activity

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Other classes of cleaving

ribozymes do not require metal ion

for activity. Amino group ofnearby nucleotide base destabilizes

the phosphodiester bond instead

Cyt

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Metabolite-responsive ribozyme-mRNA hybride

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Ligating ribozymes

Reaction mechanism similar to that of RNA polymerases, requires

Mg ion for catalysis

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RNAse P

RNAse Pcleavage

site

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RNAse P is a ribozyme

RNAse P cleaves the 5 end of pre-tRNAs

It is composed of 12 kDa P protein and about 400

nt long RNA The catalytic activity lies entirely within RNA part

Enzyme is efficient without P protein but in highsalt conditions

P protein or high salt is thought to screen therepulsive electrostatic interactions betweenRNAse P RNA and substrate pre-tRNA

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- Synthetic RNA molecule,

capable to acquire 2

completely different

secondary structures

- Each structure performs

different enzymatic

activity: ligation versus

cleavage

- Based on two different

initial ribozymes with

similar length

One sequence two ribozymes

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RNA interference (RNAi)

A natural biological mechanism for silencing genes

Revolutionary new technology (potent and simple) to

knock down gene expression in eukaryotic cells

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0

500

1000

15002000

RNAiarticleamount

1998 1999 2000 2001 2002 2003 2004year

RNA interference (RNAi)

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How was RNAi discovered?

The injection of double-stranded RNAs into C.

elegans resulted in the silencing of a gene

complementary to dsRNAs.

A- negative control (without hybridization

probe)

B- normal pattern of endogenous mex-3 RNAC- injected with antisense RNA

D- injected with dsRNA

So how does this silencing process work?

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So how does this silencing process work?

RISC - RNA induced

silencing complex

siRNA

silencing RNA

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RNAi is widespread among eukaryotes

Highly evolutionarily conserved property

Must have important functions!

Defense mechanism against

dsRNA-containing viruses

May stabilize the genome by

sequestering repetitive sequencessuch as mobile genetic elements

Control cellular development

Dicer knockout mice dont survive

past gastrulation

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RNAi technology limitations in mammalian systems

dsRNA ( >30 nt )

general

interferon response

global inhibition of

mRNA translation

chemically synthesized siRNA

cleaved by Dicer in vitro

transcribed dsRNA

effective but transient silencing of

gene expression

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Fungi, plants and worms Drosophila and mammals

cell autonomous silencing

non heritable

no indication of siRNA replication

RNAi in :

systemic nature of

silencing

heritable

can replicate siRNA

with RNA-dependent

RNA polymerases

siRNA- mediated RNAi is transient

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RNAi versus miRNA translational repression

micro RNAs (miRNAs) arenot perfectlycomplimentary to theirtargets

miRNAs do not inducetarget cleavage but blocktranslation by binding tocomplementary mRNAs

miRNAs are encoded by thehost genome, whereassiRNAs in most casesoriginate from outer source

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Is RNAi exclusively limited to cytoplasm and post-

transcriptional control ?

Although this is a very common view, it

does not always have to be the case siRNA can be transported to nucleus and act

as shRNA to block transcription

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DNA and RNA recognition models of shRNA

initiated chromatin condensation

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The RNA world did it exist?

Probably, yes

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The modern world The RNA world

DNA

RNA

Proteins

RNA

information flow

Information carryer replication

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The main requirement of RNA

world... If there was an RNA world, there must have

been an RNA molecule which is itself

capable of making RNA, or in other words an RNA ploymerase, made of RNA

So far, such a primordial polymerase is not

known to exist in nature However a synthetical RNA molecule,

capable to replicate RNA has been made

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Isolated from a pool of about 1015 synthetic RNAs, based on

ligating ribozyme

Fidelity of 96.7 %

Extension time: 14 nucleotides in 24 hours

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Late RNA world

1) ribozymes, able to catalyze peptide bond

formation and other chemical reactionsemerged. Such ribozymes have been made

in vitro.

2) proteins began to take over the enzymaticactivities

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The pre-RNA world

The available synthetic ribopolymerase is

165 nt long. Even one tenth of that is far too

long to emerge accidentally in the prebioticsoup

Some researchers argue that some sort of

yet unknown simpler polymer must haveexisted before RNA