Non-coding RNAs or RNAs come more than in three flavours...
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Transcript of Non-coding RNAs or RNAs come more than in three flavours...
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