Model of a transcription unit
RNA
polymerase 3. Termination
2. Elongation
1. Bind to promoter
DNA double helix
Attached RNA transcript
Transcription unit
Start signal Termination signal
6. Regulation of gene expression in eukaryotes
nuclear receptors (cell signaling)
Regulation is
mediated by
1) interactions of
regulatory proteins
with regulatory
sequences on DNA
2) ncRNA
Six steps of information transfer in eukaryotes
that constitute potential regulatory points of gene expression
Plasma membrane
DNA
Primary RNA
transcript
mRNA
Transcriptional
control
mRNA
Interactive mRNAProtein
Active/
Inactive
Protein
Processing
transcript
Nuclear
envelope
Transport
control
mRNA
stability
control
Translational
control by
ribosome
selection
Protein
posttranslation
control
1
2
3
45
6
Copyright © mot5111318
Levels of gene expression control
0. Chromatin
1. Where and how often is a given gene transcribed
(transcriptional control)
2. How the primary transcript is spliced (posttranscriptional-spliced
control)
3. Selection of RNAs to be transported from the nucleus to
the cytoplasm (control of RNA transport)
4. Selection of mRNAs to be translated on ribosomes
(translational control)
5. Selective destabilization of certain mRNAs in the
cytoplasm (mRNA degradation)
6. Selective activation, inactivation and
compartmentalization of specific proteins after they have
been synthesized (protein activity control - posttranslational
control, transport)
DNA segments that can modulate transcription by binding gene regulatory proteins
Upstream
enhancer
Downstream
enhancer
Core
promoter
Gene sequence
CAAT
TATA
30 bp
~ 80 bp
+1 transcription
Regulatory protein
DNA binding site
Polymerase
Unmasked DNA sites
Protein/protein interactions
+1
+1
+1
A
B
Copyright © motifolio.com5111320
Regulation is
mediated by
1) interactions of
regulatory
proteins
with regulatory
sequences on DNA
2) ncRNA
Protein-DNA interactions
- proteins interact with sugar phosphate
skeleton (phosphate) or through grooves
with
bases
- Sequences not sequence specific
(skeleton - histones; structurally specific
- HMG proteins) or sequentially
specific (skeleton + grooves combination:
BglII (AGATCT) and BamHI
(GGATCC) contact the same bases and
They "read" the curvature of the
surroundings
The shape and charge specificity of DNA
determines the types of DNA binding
domain DNA…)
Types of interactions
Phosphates can interact with salt
bridges
• Arg and Lys - saline
• salt bridges
• (positive charges Arg and Lys
• creates a bond with the negative
• phosphate group charge)
• Electrostatic charge / surface
indicates protein binding capabilities
• salt bridges - between
phosphates and + charged AK
side chains (Lys, Arg, His)
• hydrogen bonds - between
phosphates, sugars, bases in
NK and peptide bond or
hydrophilic AK side chains
• stacking - between aromatic
amino acids (Trp, Tyr, Phe, His)
and bases
• hydrophobic interactions between
bases in NK and nonpolar
side chains of AK
Hydrogen bonds
• sequence-specific protein contacts the base
("direct"
• readout) - through a large or small groove - a large
groove is
• more accessible - hydrogen bonds (donor vs
electron acceptor
related to recognition and helium - pre
interaction and helix with DNA ex. many
direct interactions between AK side chains
and NA bases.
Uncommon interactions:
O6 or N7 guan atoms…
Side chains Arg, Lys, Gln, Asn, Ser
Less often:
N6 or N7 adenine atoms
Exceptionally:
Pyrimidines
often occurs: more H-bonds to 1 base
water-mediated H-bond between protein
and NA
Binding of proteins to DNA via hydrogen bonds
• The large groove has the size corresponding to the dimensions and the helix
• and has exposed H-linking groups
• Ade residues C-6 (NH2) and N-7 may form specific ones
• hydrogen bonds with Gln and Asn
• Gua can form specific hydrogen bonds with Arg
• Strong binding, sequence specific - affinity nM - uM
• Weak binding, structural specific - affinity uM - mM
Protein motifs interacting with DNA
Rooman, Marianne and Wintjens,
René (March 2015)
Protein–DNA Interactions. In: eLS.
John Wiley & Sons, Ltd:
Chichester.
DOI:
10.1002/9780470015902.a0001348.
pub3
Motivy- Transkripční faktory –zipper typ
GCN4 a AP1 a c-Myc
Helix-otáčka-helix
– HTH
– Winged helix
– TALE
General transcription
factors-motive HTH
Zinc finger
DNA-interacting p53 protein
Rooman, Marianne and Wintjens, René
(March 2015)
Protein–DNA Interactions. In: eLS. John Wiley
& Sons, Ltd:
Chichester.
DOI:
10.1002/9780470015902.a0001348.pub3
Loop-sheet-helix
- loops coming out of
main core domain - protrudes
b-sheet and α-helix
- 3 Cys and 1His coordinate Zn
helix in a large groove and
loop in a small groove
- Activation of transcription through
acidic TA domain
- core / DNA-binding domain p53 transcription
factor important
for cell cycle regulation, apoptosis and repair
of the damaged
DNA (tumor suppressor)
Motives receptors for hormons,loop-sheet-helix, GAL4
15
Regulation at the transcriptional level
Basic regulation of transcription
(common to all genes)
Regulation by components of the " basal
transcription complex " (RNA polymerase binding to
the TATA box, TATA binding proteins and other
"basal" transcription factors binding to the RNA
polymerase or in the promoter region)
Genes regulated only in this way:
Constitutively expressed genes
Specific effects on gene expression:
Through regulatory sequences in DNA
and specific transcription factors.
basal transcription complex
Scheme of the activity of a master gene
Master gene regulatory protein
mRNA mRNA
Protein 1 Protein 3
Gene 1 Gene 2 Gene 3 Gene 4
On Off On Off
No protein 2 No protein 4
Binding to
DNA elements
Copyright © motifolio.com5111321
17
transcription factors
Animal Genetics - Gene exoression. [online]. [cit. 2014-08-15]. Dostupné z:
http://web2.mendelu.cz/af_291_projekty2/vseo/stranka.php?kod=307
Necessary to initiate transcription
They usually induce transcription, exceptionally they can
inhibit it
Their various combinations bind to the promoter before
the RNA polymerase is attached
General transcription factors
in all or most cell types
necessary to induce transcription
basal TF-low activity, minimal cell requirements
most common: TFIIA, TFIIB, TFIID (includes a subunit
called TATA binding protein (TBP) - binds specifically to
the TATA box sequence), TFIIE, TFIIF and TFIIH)
constitutive TF - increase the basal activity of the cell
according to the cell type, the basic requirements of the
cell (present (and active) in the cell at all times
- general transcription factors, Sp1, NF1, CCAAT)
- special transcription factors
They apply to inducible transcription
- only in cells of certain tissues and certain situations
(example p53)
https://employees.csbsju.edu/hjakubowski/classes/ch331/bind/eukar
ypromNat0703e.htm
Figure 1 Comparison of a simple eukaryotic promoter and extensively
diversified metazoan regulatory modules. a, Simple eukaryotic
transcriptional unit. A simple core promoter (TATA),
upstream activator sequence (UAS) and silencer
element spaced within 100–200 bp of the TATA box
that is typically found in unicellular eukaryotes. b,
Complex metazoan transcriptional control modules. A complex
arrangement of multiple clustered enhancer modules interspersed with
silencer and insulator elements which can be located 10–50 kb either
upstream or downstream of a composite core promoter containing TATA
box (TATA), Initiator sequences (INR), and downstream promoter
elements (DPE).
18
Terminology
Enhancers - regulatory sequences in
DNA that bind transactivators
Transactivators bind coactivators
Silencers - regulatory sequences
that bind the corepressor
Hormones bind to the intracellular
receptor, which binds to the hormone
response element
These terms are still used. The
terms are gradually being replaced:
regulatory sequences in DNA
(enhancer, silencer, hormone
response element)
specific transcription factors
(different from basal transcription
factors)
mediator proteins -coactivators
•Transcription factors are proteins that help turn specific genes "on" or
"off" by binding to nearby DNA.
•Transcription factors that are activators boost a gene's transcription.
Repressors decrease transcription.
•Groups of transcription factor binding sites called enhancers and
silencers can turn a gene on/off in specific parts of the body.
•Transcription factors allow cells to perform logic operations and combine
different sources of information to "decide" whether to express a gene.
➢Components of the
eukaryotic promoter:
5_regulace_2020FaF 19
http://www.cbs.dt
u.dk/dtucourse/co
okbooks/dave/Lek
t03bkg.html
• Basal transcription
factors
(basal, constitutive)
Special transcription
factors
1.Constitutive - present
(and active) in the cell at
all times - general
transcription factors,
Sp1, NF1, CCAAT
2.Conditionally active their
activation required
https://employees.csbsju.edu/hjakubowski/classes/ch331/bind/olbindtransciption.html
5_regulace_2020FaF 20
Mechanical division
Transcription factors of the general
transcription complex (TFIIA, TFIIB,
TFIID, TFIIE, TFIIF, TFIIH - are
ubiquitous and react with the
promoter (often TATA box) of
structural genes, important in the
development of vertebrates and
invertebrates
Upstream transcription factors (UTF) upstream
- towards the 5´ part,
proteins that bind to the regulatory
part of the RNA polymerase I
promoter at position -110 to -180, the
presence is not necessary to initiate
transcription, but multiplies its
efficiency (it can also repressive)
Inducible transcription factors - same
as UTF, but need to be activated or
inhibited
5_regulace_2020FaF 21
A. “The eukaryotic transcription apparatus can
be divided into three sets, which include the
RNA polymerase II core complex and related
general transcription factors (TFIIA, -B, -D, -E,
-F and -H), multi-subunit cofactors (mediator,
CRSP, TRAP, ARC / DRIP, etc.) and various
chromatin modifying or remodeling
complexes (SWI / SNF, PBAF, ACF, NURF and
RSF).
B. Metazoa organisms have developed multiple
gene-selective and tissue-specific TFIID-like
assemblies using alternative TAFs (TBP [TATA
Binding Protein] -related factors such as
ovarian-specific TAF105), as well as TRF (TBP[TATA
Binding Protein-associated factors] related
factors, such as is TRF2 in Drosophila
and mice), which mediate the formation of
specialized RNA polymerase initiation
complexes that direct the transcription of
tissue-specific and gene-selective expression
programs. "(Natural link in the picture
above.)""
➢Methods of activation of transcription
factors:
➢ligand-induced conformation change (signal, eg
hormone)
➢conformational change after removal of the
inhibitory protein
➢conformational transition induced by
phosphorylation
➢phosphorylation by protein kinase
➢phosphatase dephosphorylation
➢stabilization of the active conformation of the
transcription factor against its degradation
5_regulace_2020FaF 22
Gene regulation by members of the nuclear receptor superfamily
A. Glucocorticoid receptor
Cortisol
Hsp70
Hsp90
Inactive receptor IP
Transcription activated
Plasma membrane
Nucleus
Copyright © motifolio.com5111362
The glucocorticoid receptor (GR, or GCR),
also known as NR3C1 (nuclear receptors of
subfamily 3, group C, member 1), is a receptor
to which cortisol and other glucocorticoids
bind. GR is expressed in almost every cell in
the body and regulates genes that control
development, metabolism and the immune
response. Because the receptor gene is
expressed in several forms, it has many
different (pleiotropic) effects in different parts
of the body. When glucocorticoids bind to GR,
its primary mechanism of action is the
regulation of gene transcription. The unbound
receptor resides in the cytosol of the cell. After
the receptor is bound to the glucocorticoid, the
receptor-glucocorticoid complex can proceed
in either of two ways. The activated GR
complex regulates the expression of antiinflammatory
proteins in the nucleus or
suppresses the expression of proinflammatory
proteins in the cytosol (by
preventing the translocation of other
transcription factors from the cytosol to the
nucleus). In humans, the GR protein is
encoded by the NR3C1 gene, which is located
on chromosome 5 (5q31). Glucocorticoid
receptor -
https://en.qaz.wiki/wiki/Glucocorticoid_recepto
r
Gene regulation by members of the nuclear receptor superfamily
B. Estrogen receptor
Transcription activated
Estrogen
Receptor dimer
Hsp90
Inactive receptor
HAT
Coactivator
Plasma membrane
Nucleus
HAT – histone acetyltransferase
Copyright © motifolio.com
.[2] Estrogen receptors (ERs) are steroid receptors
present in the cell nucleus [1] of vertebrates to
which estrogen binds. Humans and other
mammals have two types of estrogen receptors,
the estrogen receptor α (ERα, also ESR1) and the
estrogen receptor β (ERβ, also ESR2). Both
receptors can form homodimers as well as
common heterodimers. However, the GPER
receptor, which is a special G protein-coupled
receptor, also responds to estrogen. [2] All of these
types of receptors also occur in other vertebrates,
including fish. [2]
Estrogen receptors allow the detection of estrogen at specific sites in
the vertebrate body. At rest, they are usually found in the cytosol, while
upon binding to the ligand (estrogen), they are activated, dimerized,
and enter the cell nucleus. There it binds to DNA sequences known as
estrogen responsive units (EREs). The binding is also affected by other
co-regulators (coactivators and corepressors). [3]
Due to its receptors, estrogen controls reproduction, both the
development of the reproductive system and reproductive behavior. The
best known, however, is the influence on the development of female
(female) genitals. Furthermore has several functions not related to
reproduction, e.g. affects bone density and strength, blood lipid levels,
fat storage, and management of water with salts, as well as some
higher brain functions (memory effect). However, it probably also affects
the development of parts of the male reproductive system, such as
sperm maturation. [2]
Gene regulation by members of the nuclear receptor superfamily
C. Thyroid receptor
Transcription activated
HAT
Coactivator
HDAC
Thyroid
Plasma membrane
Nucleus
Absence of hormone
Corepressor
Thyroid hormone
receptor
Transcription
repressed
HDAC – histone deacetylase
HAT – histone acetyltransferase
Copyright © motifolio.com
5111364
The thyroid hormone receptor (TR)[1] is a
type of nuclear receptor that is activated by
binding thyroid hormone.[2] TRs act as
transcription factors, ultimately affecting the
regulation of gene transcription and
translation. These receptors also have nongenomic
effects that lead to second
messenger activation, and corresponding
cellular response.[3]
Thyroid hormone receptors regulate
gene expression by binding to
hormone response elements (HREs)
in DNA either as monomers,
heterodimers with other nuclear
receptors, or homodimers.[4]
Dimerizing with different nuclear
receptors leads to the regulation of
different genes. THR commonly
interacts with the retinoid X receptor
(RXR), a nuclear retinoic acid
receptor.[9] TR/RXR heterodimers are
the most transcriptionally active form
of TR.[10]
Regulation after transcription
Alternative splicing, miRNAs and siRNAs, translation initiation factors, & protein
modifications.
• Even after a gene has been
transcribed, gene expression
can still be regulated at various
stages.
• Some transcripts can undergo
alternative splicing, making
different mRNAs and proteins
from the same RNA transcript.
• Some mRNAs are targeted by
microRNAs, small regulator
RNAs that can cause an mRNA
to be chopped up or block
translation.
• A protein's activity may be
regulated after translation, for
example, through removal of
amino acids or addition of
chemical groups.
5_regulace_2020FaF 26
Regulation of RNA level
rare in bacteria, common in higher organisms
RNA processing- alternative splicing
mRNA stability - for many genes, RNA interference
affects life span or translation rate
translation - regulatory proteins bind to mRNA and /
or the ribosome and affect the translation rate
control options:
mRNA degradation rate control (mRNA stability)
converting the non-translatable mRNA into a form
that can be translated
translational control by regulatory proteins
binding of antisense RNA to mRNA
27
Regulation of gene expression by transcriptional modification
Alternative splicing and variation
of thepolyadenylation site at the 3
'end causes a single gene to
produce different proteins
RNA editing
In some cases, the RNA may be
edited after transcription.
The primary transcript (hnRNA)
is identical, after transcription
there is a base exchange or
nucleotide addition (deletion)
Gen apoB produkuje v játrech protein obsahující 4563 AK
Tentýž gen v enterocytech produkuje apoB obsahující jen 2152 AK
Konverze C(cytosin) na U (uracil) deaminací v RNA transkriptu generuje stop-kodón v intestinální
mRNA. Tak protein produkovaný v enterocytu má pouze 48 % délky proteinu hepatálního
Syntéza apoB v hepatocytech a
enterocytech (je součástí
chylomikronů a VLDL)
RNA level regulation
• RNA stability
• mRNA has a short
half-life, upon
degradation it
undergoes
ribonuclease
degradation
• sensitivity to
RNases depends on
the secondary
structure
• this may be
affected by
regulatory signals
that induce the
binding of
regulatory proteins
to RNA
5_regulace_2020FaF 28
• Translation
regulation
• the ribosome
binding site
(RBS) on the
mRNA may be
hidden by the
secondary
structure
• cleavage of a
portion of the
mRNA by
RNase III
restores RBS
accessibility
RNA stability
- mRNA has a short half-life, it is
readily degraded by ribonucleases
- mRNA secondary structure is a key
component in RNAse sensitivity
- mRNA secondary structure can be
altered by protein binding –
regulation signals
8_MB-2017
General scheme of messenger RNA
decay pathways.
8_MB-2017
(A) mRNAs containing an AU-rich element
(ARE) in their 3' UTR undergo rapid AREmediated
mRNA decay (AMD) in resting
cells. Concealing ARE sequence from AMD
induces gene expression. (B) Quality
control mechanisms. mRNAs that contain a
premature termination codon (PTC) are
recognized and specifically degraded by the
nonsense-mediated mRNA decay (NMD)
pathway. (C) The basic mRNA decay
machinery in the cytoplasm initially
removes the poly(A) tail through the
activity of deadenylating enzymes.
Subsequently, the mRNA can be further
degraded from the 3' end by a complex of
3'–5' exonucleases known as the exosome.
Alternatively, the mRNA is decapped at the
5' end, and the 5'–3' exonuclease Xrn1
proceeds to degrade the body of the
mRNA.
3'-5' decay
5_regulace_2020FaF 31
TEST
Regulatory mechanisms mediated
by transcription factors
by RNA
8_MB-2017
Functional types of RNA
hnRNA
rRNA
tRNA
snRNA, snoRNA, …
mRNA
siRNA, miRNA, …
DNA
pre-rRNA
pre-tRNA
Translation; encodes a sequence of proteins
Translation; part of the ribosome
Translation; transfer / amino acids activation
Splicing / modification
of RNA
Regulation of gene
expression
Small non-coding RNAs
Long non-coding RNAs
TEST 8_MB-2017
encoding genes represent less than 2% of the total genome sequence
vs.
at least 90% of the human genome is actively transcribed
the more complex organism, the more it comprises non-coding RNAs
World of noncoding RNAs
Recent evidence suggests that the non-coding RNAs (ncRNAs) may play
major biological roles in cellular development, physiology and pathologies.
NcRNAs could be grouped into two major classes based on the transcript
size: small ncRNAs and long ncRNAs.
The percentage of
protein-coding
genes sequences
in several
eukaryotic and
bacterial genomes.
8_MB-2017
Small non-coding RNAs
miRNA
siRNA
piRNA
snoRNA
PARS
tiRNA
Long non-coding RNAs
lincRNA
TERRAs
T-UCR
microRNA (miRNA)
Piwi-interacting RNA (piRNA)
small interfering RNA (siRNA)
small nucleolar RNA (snoRNAs)
tRNA-derived small RNA (tsRNA)
small rDNA-derived RNA (srRNA)
small nuclear RNA,
also commonly referred to as U-RNA
8_MB-2017
RNA interference - RNAi
• sequence-specific gene silencing mechanism triggered
by double stranded RNA, on the post-transcriptional
level or transcriptional level
- inhibitory elements are small RNA molecules (miRNAs,
siRNAs….)
- miRNAs generated by cleavage of larger pre-miRNA
molecules
• nucleases Drosha and DICER, which are compiled into
multiprotein complex RISC (RNA-induced silencing
complex) with proteins Argonaut
• RNA interference is a process by which noncoding RNA
molecules interfere (pair) with target regions of mRNA,
resulting in prevention of gene expression of these
mRNAs.
• For short, this proces is also called RNAi. We rank him
among posttransriptional mechanisms of gene
expression.
• Most eucaryotic organisms is capable of RNA
interference, the process was first studied in the C.
elegans.
Pri-
miRNA
8_MB-2017
RNA interference
RISC has helicase activity, thanks to
which miRNA is loosened; only one chain
remains associated with the complex
that allows sequence-specific binding of
the whole complex to the target
complementary mRNA
nuclease activity of RISC complex
cleaves the mRNA - its degradation
occurs
Originally protecting cells against
viruses
common in eukaryotic cells
useful for targeted inactivation of
genes: research of gene functions
8_MB-2017
Cenorhabditis
elegans
Discovery of RNA interference
(1998)
- silencing of gene expression with dsRNA
8_MB-2017
Mechanism of action of small RNAdepends
on the length of sRNA, biogenesis (precursor),
...
PTGS (posttranscriptional gene silencing ):
- specific transcription degradation or translation blocking
TGS (transcriptional gene silencing ):
- methylation of cytosines in the promoter (RdDM),
heterochromatinization, inhibition of transcription factor binding
Pol VPol II
TGS
PTGS
PTGS
TEST 8_MB-2017 39
Basic mechanism of RNAi
dsRNA in cell is cleaved by
RNase DICER into short
dsRNA fragments – sRNA
Argonaute with a single
strand (from sRNA)
mediates recognision of
complementary sequences,
which should be silenced
(TGS, PTGS)
8_MB-2017 40
Small RNA in plants/animals
- 3’ end of sRNA methylated (HEN1) - protection
• miRNA (micro) – from transcipts of RNA Pol II (pre-miRNA)
– hunderds MIR genes (in trans)
Pol II
DROSHA (Rnaze III),
PASHA (RNA binding protein),
DICER
• siRNA (small interfering) – from dsRNA of various origin (both internal and
external – thousands types (both in cis and in trans)
….. (+ piRNA in animals)
pre-miRNA
Wang et al. 2004
8_MB-2017 41
miRNA biogenesis
8_MB-2017 42
8_MB-2017 43
siRNA
Dicer, also known as endoribonuclease Dicer or helicase with RNase
motif, is an enzyme that in humans is encoded by the DICER1 gene. Being
part of the RNase III family, Dicer cleaves double-stranded RNA (dsRNA)
and pre-microRNA (pre-miRNA) into short double-stranded RNA fragments
called small interfering RNA and microRNA, respectively. These fragments
are approximately 20-25 base pairs long with a two-base overhang on the
3' end. Dicer facilitates the activation of the RNA-induced silencing
complex (RISC), which is essential for RNA interference. RISC has a
catalytic component argonaute, which is an endonuclease capable of
degrading messenger RNA (mRNA).
• siRNA (small interfering) from dsRNA of various origin (both internal and
external – thousands types (both in cis and in trans)
….. (+ piRNA in
animals)
DICER
Argonaute
RNA binding protein (20-26 nt RNA)
- strand selection (5’ nt,
participation of HSP90)
- 10 genes in Arabidopsis
- main component of RISC
(RNA induced silencing complex)
- block of translation or slicer
(RNAse H-like endonuclease
- PIWI doména)
- role in TGS (RdDM)
(RNA directed DNA methylation)
8_MB-2017 44
Mechanism of small RNA action - overview
PTGS (21-22 nt): - specific cleavage of transcript
- - block of translation
TGS (24 nt): - methylation of promoter, heterochromatin formation
- preventing interaction of transcription factors
Pol V
8_MB-2017
sRNA mode of action also depends
on complementarity
- incomplementarity in cleavage site prevents RNase activity
8_MB-2017
dsRNA formation
(MIR genes)
?
• RdRP = RNA-dependent RNA Polymerase – synthesis of compl. RNA strand
templates: - transcripts cleaved by RISC
- impaired mRNAs (without polyA or cap)
- transcripts of RNA polymerase IV
+ secondary structures of viral RNAs (!)
mRNA fragment after Ago
cleavage (secondarysiRNA )
8_MB-2017 47
8_MB-2017 48
Matzke MA, Matzke AJM – This figure is adapted from one by Matzke MA, Matzke AJM (2004) Planting the Seeds of a New
Paradigm. PLoS Biol 2(5): e133 doi:10.1371/journal.pbio.0020133.
Overview of RNA interference. The
dicer enzymes produce siRNA from
double-stranded RNA and mature
miRNA from precursor miRNA. miRNA
or siRNA is bound to an argonaute
enzyme and an effector complex is
formed, either a RISC (RNA-induced
silencing complex) or RITS (RNAinduced
transcriptional silencing)
complex. RITS affects the rate of
transcription by histone and DNA
methylation, whereas RISC degrades
mRNA to prevent it from being
translated.
Overview of RNA interference
microRNA
5_regulace_2020FaF 49
small non-coding RNA of 18-25 nucleotides in size
negative regulatory expression genes that
degrade target mRNA or block its translation
microRNAs arise from primary pri-miRNA
transcripts that are relatively large (even several
kb)
pri-miRNAs are treated in the nucleus with
Drosha RNAase and protein
Pasha binding dsRNA to pre-miRNA about 70
nucleotides long with imperfect hair structure
pre-miRNAs are exported to the cytoplasm by
Exportin 5 and digested with Dicer nuclease to
final 22 kb miRNA duplexes
The miRNA binds to the RISC, one fiber degrades
and the other mediates the degradation or
translation inhibition of the respective mRNA
8_MB-2017
RNA
interference
based on enzyme
degradation or
translation
inhibition of
specific mRNA
Drosha (RnasaIII)
Pasha (protein)
Exportin 5 (transporter)
Dicer (RNasaIII)
RISC (multiprotein
complex)
TEST
8_MB-2017
siRNA and miRNA utilisation:
➢ iRNA usage does not fall under GMO
➢ Yet usage of cassettes producing iRNA
does!
1) gene analysis
2) gene therapies
3) anti-viral vaccines
4) transgenic organisms that have
transiently inhibited selected genes
8_MB-2017
MicroRNAs as tumor suppressors or oncogenes
8_MB-2017
CRISPR system
In 2008, it was described RNAi analogous system
designed to the degradation of viral NA
➢It uses internal "virus" sequences inserted in the inverted repeats
(CRISPR)
➢CRISPR = clusters of regularly interspaced short
palindromic repeats
➢After transcription of this sequence leads to their progressive cleavage
by Cas proteins
➢The resulting products interfere with the nucleic acid of the entering
virus
• Each of repeats followed by short segments called Spacer DNA, obtained during
previous meetings with relevant bacterial viruses or plasmids.
Brouns et al. (2008): Small CRISPR RNAs Guide Antiviral Defense
in Prokaryotes, Science 321, 960-964
8_MB-2017
Horvath, 2010
Science
Overview of the CRISPR/Cas
mechanism of action. (A)
Immunization process: After
insertion of exogenous DNA
from viruses or plasmids, a Cas
complex recognizes foreign
DNA and integrates a novel
repeat-spacer unit at the leader
end of the CRISPR locus. (B)
Immunity process: The CRISPR
repeat-spacer array is
transcribed into a pre-crRNA
that is processed into mature
crRNAs, which are
subsequently used as a guide
by a Cas complex to interfere
with the corresponding
invading nucleic acid. Repeats
are represented as diamonds,
spacers as rectangles, and the
CRISPR leader is labeled L.
8_MB-2017
CRISPR/C
as9
• the whole system is modified for targeted
mutagenesis
• - vector - gRNA = crRNA tracr + RNA
•
• part gRNA and 20nt complementary
section to the target site in the genomic
DNA
• - + Coexpression of Cas9 nuclease (even
the same
vector)
8_MB-2017
• PAM – protospacer adjacent motif
• sequence in the vicinity of gDNA
• required for efficient cleavage by Cas9 nuclease
• the original system "NGG" (but the development of systems with other
sequences)
• according to the system target sequence must be in the N 20 -GG
8_MB-2017
gDNA libraries available for various
organisms
- beware of non-specific binding - nonspecific
cleavage
- use several different gDNA
- reversion of the phenotype by
increasing the expression
- introduction of mutation
- Various companies - different
platforms (modifications of the original
system)
- available software can be used to
design
A. Wild-type Cas9 nuclease site specifically cleaves double-stranded DNA activating double-strand break repair
machinery. In the absence of a homologous repair template non-homologous end joining can result in indels
disrupting the target sequence. Alternatively, precise mutations and knock-ins can be made by providing a
homologous repair template and exploiting the homology directed repair pathway.
B. Mutated Cas9 makes a site specific single-strand nick. Two sgRNA can be used to introduce a staggered
double-stranded break which can then undergo homology directed repair.
C. Nuclease-deficient Cas9 can be fused with various effector domains allowing specific localization. For
example, transcriptional activators, repressors, and fluorescent proteins.
8_MB-2017
2009
lncRNA – long non-coding RNA
lncRNA jsou předpovězené
B-Okazakiho fragmenty
8_MB-2017
Long non-coding RNAs
(long ncRNAs, lncRNA)
are non-protein coding
transcripts longer than
200 nucleotides.[1] This
somewhat arbitrary limit
distinguishes long
ncRNAs from small
regulatory RNAs such as
microRNAs (miRNAs),
short interfering RNAs
(siRNAs), Piwiinteracting
RNAs
(piRNAs), small nucleolar
RNAs (snoRNAs), and
other short RNAs.[2]
Long ncRNAs in the regulation of gene transcription
8_MB-2017
In eukaryotes, RNA transcription is a
tightly regulated process. NcRNAs can
target different aspects of this process,
targeting transcriptional activators or
repressors, different components of the
transcription reaction including RNA
polymerase (RNAP) II and even the DNA
duplex to regulate gene transcription and
expression (Goodrich 2006). In
combination these ncRNAs may comprise
a regulatory network that, including
transcription factors, finely control gene
expression in complex eukaryotes.
Long ncRNAs in genespecific
transcription