Non-coding RNAs

or RNAs come more than in three flavours...

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

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

Housekeeping ncRNAs

• tRNA and rRNA - translation

• snRNA – Pre-mRNA splicing

• snoRNA – rRNA modification

• gRNA – guide RNA in RNA editing

• Telomerase RNA – primer for telomeric DNA synhesis

• A few other...

4.5S RNA and 7S RNA – a part of signal recognition particle (SRP)

• SRP recognizes signalling amino acid sequence in the N-terminus of growing polypeptide chain

• Upon signal recognition, ribosome is attached to endoplasmatic reticlum so that the protein, made by ribosome, enters the secretory pathway

ribosome

Signal sequence

Endoplasmatic reticlum membrane

mRNASRP

translocon SRP

SRP receptor

Structure of SRP

RNA holds together the protein subunits of SRP as well as helps to bind to ribosome

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

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

• The tRNA part of tmRNA (black) adds an alanine to the growing 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

Regulatory ncRNAs

• Transcriptional regulators

• Translational regulators

• Modulators of protein function

• Regulators of RNA and protein distribution

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 dosage compensation

Dosage compensation mechanisms

The role of roX ncRNAs in dosage compensation in Drosophila

MSL-1

MSL-3

MOF histone acetylase

MLEhelicase

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 H4 histones 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

Acetylated lysines

Silencing of one female X chromosome in mammals

• The X chromosome silencing is mediated by Xist – a 16,000 nt long 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 is able to regulate Xist activity by base-pairing to it

Genetic imprinting and shRNAs• Genetic imprinting is a process which results in expression on

only one allele of gene, while the allele originating from the other parent is silenced

• Process is somewhat similar to dosage compensation• The differences of expression from both alleles are due to

different states of chromatin (euchromatin and heterochromatin) and also to differential methylation of DNA

• Activity of small heterochromatic RNAs (shRNAs) appear to be essential for establishing and maintaining the imprinted status of genes

• Activity of various shRNAs is not limited only to genetic imprinting

DNA and RNA recognition models of shRNA initiated chromatin condensation

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

Translation of human HFE gene is downregulated by anti-sense RNA

HFE promoter

Anti-sense promoter

Sense exons

Anti-sense exons

Ribosome

HFE mRNAA

Anti-sense RNA

B

DsrA RNA in E.coli activates ribosome binding to stress-response factor rpoS mRNA

Ribosome binding site blocked by base-pairing

rpoS mRNA

DsrA RNA

RBS accessible

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 transcription factors

6S RNA modulates 70 function in E.coli

+ + +70 RNA pol

6S RNA

Log-phase Stationary phase

Ribozymes• RNA molecules with catalytical properties (Ribonucleic acid

enzymes) • In nature ribozymes occur mostly within self-splicing intrones and

RNA 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 other

reactions than RNA cleavage and ligation

Cleaving ribozymes

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

The 3D structure of hammerhead ribozyme

Hammerhead ribozyme mechanism

Requires bivalent metal ion for activity

Other classes of cleaving ribozymes do not require metal ion for activity. Amino group of nearby nucleotide base destabilizes the phosphodiester bond instead

Cyt

Metabolite-responsive ribozyme-mRNA hybride

Ligating ribozymes

Reaction mechanism similar to that of RNA polymerases, requires Mg ion for catalysis

RNAse P

RNAse P cleavage site

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 high

salt conditions• P protein or high salt is thought to screen the

repulsive electrostatic interactions between RNAse P RNA and substrate pre-tRNA

- 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

RNA interference (RNAi)

A natural biological mechanism for silencing genes

Revolutionary new technology (potent and simple) toknock down gene expression in eukaryotic cells

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RNA interference (RNAi)

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 RNAD- injected with dsRNA

So how does this silencing process work?

RISC - RNA inducedsilencing complex

siRNA – silencing RNA

RNAi is widespread among eukaryotes

Highly evolutionarily conserved property

Must have important functions!

Defense mechanism againstdsRNA-containing viruses

May stabilize the genome by sequestering repetitive sequences such as mobile genetic elements

Control cellular developmentDicer knockout mice don’t survivepast gastrulation

RNAi technology limitations in mammalian systems

dsRNA ( >30 nt )

generalinterferon response

global inhibition ofmRNA translation

chemically synthesized siRNA

cleaved by Dicer in vitrotranscribed dsRNA

effective but transient silencing of gene expression

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

RNAi versus miRNA translational repression

• micro RNAs (miRNAs) are not perfectly complimentary to their targets

• miRNAs do not induce target cleavage but block translation by binding to complementary mRNAs

• miRNAs are encoded by the host genome, whereas siRNAs in most cases originate from outer source

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

DNA and RNA recognition models of shRNA initiated chromatin condensation

The RNA world – did it exist?

• Probably, yes

The modern world The RNA world

DNA

RNA

Proteins

RNA

information flow

Information carryer replication

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

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

• Fidelity of 96.7 %

• Extension time: 14 nucleotides in 24 hours

Late RNA world

• 1) ribozymes, able to catalyze peptide bond formation and other chemical reactions emerged. Such ribozymes have been made in vitro.

• 2) proteins began to take over the enzymatic activities

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 prebiotic soup

• Some researchers argue that some sort of yet unknown simpler polymer must have existed before RNA