Transcript Non-coding RNAs - Uppsala University

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)
Signal
sequence
SRP
ribosome
mRNA
SRP
receptor
translocon
SRP
Endoplasmatic reticlum membrane
• 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
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
Ala
3’
• 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 Cterminus
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
MLE
helicase
MSL-2
MSL-1
MOF
histone
acetylase
MSL-3
2 x MSL-3
roX1/roX2
ncRNAs
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
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
Sense exons
Anti-sense
exons
Anti-sense
promoter
HFE mRNA
A
B
Ribosome
Anti-sense RNA
DsrA RNA in E.coli activates ribosome binding to
stress-response s factor rpoS mRNA
DsrA RNA
Ribosome binding site
blocked by base-pairing
rpoS mRNA
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 s70 function in E.coli
Log-phase
s70
+
RNA pol
Stationary phase
+
+
6S RNA
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
Cleavage
Y=C or T, R=A or G, H=A,T or C
Dot represents any nucleotide
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
One sequence – two ribozymes
-
-
-
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
RNA interference (RNAi)
A natural biological mechanism for silencing genes
Revolutionary new technology (potent and simple) to
knock down gene expression in eukaryotic cells
RNAi article amount
RNA interference (RNAi)
2000
1500
1000
500
0
1998 1999 2000 2001 2002 2003 2004
year
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 RNA
C- injected with antisense RNA
D- injected with dsRNA
So how does this silencing process work?
RISC - RNA induced
silencing complex
siRNA – silencing RNA
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 sequences
such as mobile genetic elements
Control cellular development
Dicer knockout mice don’t survive
past gastrulation
RNAi technology limitations in mammalian systems
dsRNA ( >30 nt )
chemically synthesized siRNA
cleaved by Dicer in vitro
transcribed dsRNA
general
interferon response
global inhibition of
mRNA translation
effective but transient silencing of
gene expression
RNAi in
Fungi, plants and worms
• systemic nature of
:
Drosophila and mammals
• cell – autonomous silencing
silencing
• heritable
• can replicate siRNA
with RNA-dependent
RNA polymerases
• non – heritable
• no indication of siRNA replication
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 posttranscriptional 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
information flow
Information carryer replication
RNA
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