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Messenger RNA maturation
Dr.ssa Mariangela Morlando
Features of RNA molecule
 Synthetised from a DNA template (transcription)
 Single strand molecule
 Contains ribose in place of deoxyribose
 Contains Uracil in place of Thymine
Types of RNA molecules
 Coding RNA which are translted into proteins: messenger
RNA(mRNA)
 Non coding RNA which are never translated into protein but
work as RNA with structural and/or regulatory function.
Messenger RNA
mRNA structures
AUG
UAA
cap
AAAAAAAAAAAAA
3’ UTR
5’ UTR
ORF
Eucaryotic gene expression
transcription
START site
exons
gene
upstream
elements
promoter
elements
TRANSCRIPTION
CAPPING
introns
pre-mRNA
m7 G
SPLICING
POLYADENYLATION
m7 G
m7 G
AAAAAAAAAn
AAAAAAAAAn
TRANSPORT
TRANSLATION
polyadenylated pre-mRNA
mature mRNA
Eucaryotic gene expression
transcription
START site
exons
gene
upstream
elements
promoter
elements
TRANSCRIPTION
CAPPING
introns
pre-mRNA
m7 G
SPLICING
POLYADENYLATION
m7 G
m7 G
AAAAAAAAAn
AAAAAAAAAn
TRANSPORT
TRANSLATION
polyadenylated pre-mRNA
mature mRNA
Why the Cap structure is important?
1) RNA stability
2) Favours the mRNA transport to the cytoplasm
3) Increases translation (it binds to eIF4E that belongs to translation
initiation complex)
7-metilguanosina
This involves the addition of a modified
guanine base to the 50 end of the RNA.
Specifically, it is a methylated guanine,
and it is joined to the RNA transcript by
an unusual 5‘–5’ linkage involving
three phosphates
•CAP is added at very early stage of
transcription initiation
estremità 5’
•The 5’-5’ phosphodiester bond makes
della RNA catena the
molecule resistant to the
exonuclease activity.
•I vitro synthetized RNA without CAP are
rapidly degraded
pre-mRNA
capping
The 5’ cap is created in
three enzymatic steps:
1.
1.
1.
a phosphate group is
removed from the 5’ end of
the transcript.
GMP moiety is added.
GMP nucleotide is modified
by the addition of a methyl
group.
RNA triphosphatase
RNA guanylyltransferase
RNA(G-7-) methyltransferase
5’ CAP favours the mRNA transport to the cytoplasm
CBC: CAP binding complex
Phosphorylation state of the CTD of RNA polymerase II along
transcription cycle
DSIF/NELF-mediated checkpoint to ensure pre-mRNA capping.
TFIIH
phosphorylate
s the RNAP II
CTD on Ser
5.
DSIF interacts
with RNAP II
shortly after
initiation.
NELF recognizes the
RNAP II–DSIF complex
and halts elongation. This
pause allows the
recruitment of the capping
enzyme (CE) which adds a
5′-cap to the nascent
transcript
NELF is released by the concerted
action of P-TEFb phosphorylation of
DSIF and the CTD on Ser 2, PRMT1/5
methylation of Spt5, and the capping
enzyme itself. FCP1 may also
participate, as FCP1 is required to
release the capping enzyme. The
precise mechanism causing NELF
release is unknown.
Robert J. Sims III et al. Genes Dev. 2004;18:2437-2468
Eucaryotic gene expression
transcription
START site
exons
gene
upstream
elements
promoter
elements
TRANSCRIPTION
CAPPING
introns
pre-mRNA
m7 G
SPLICING
POLYADENYLATION
m7 G
m7 G
AAAAAAAAAn
AAAAAAAAAn
TRANSPORT
TRANSLATION
polyadenylated pre-mRNA
mature mRNA
What is the function of the polyA tail?
1) RNA stability
2) Favours the mRNA transport to the cytoplasm
3) Increases translation efficiency by favouting the loading of
ribosomal 40S subunit
4) mRNA 3’ end formation allows efficient transcription
termination.
Looking for consenus sequences
SEQUENCE ALLINEAMENT OF cDNAs STARTING FROM THE POLYA TAIL
15-20 nt
5’UTR
globin
Ig
Ovoalbumin
Polymerase
Myc
3’UTR
AUG
AUG
UAA GGUUAUCCAUCAAUAAA…….GCUAUACGCAAAA
n
UAA CCACUGGGCCAAUAAA…….GCUAUACGCAAAA
n
AUG
UAA GCAACCUCGAAUAAA…….GCUAUACGCAAAA
n
AUG
UAA AUCUGGAGGAAUAAA…….GCUAUACGCAAAA
n
AUG
UAA UAUAGAUCCAAUAAA…….GCUAUACGCAAAA
n
AAUAAA
consensus
consensus
Polyadenylation efficiency
Influence of
consensus
sequence
AAUAAA
Efficiency
Site-specific mutation
Recombinant DNA preparation
mutazione
*
transfection
mut1 mut2 mut3 WT
polyA
HeLa cells
Northern blot
RNA extraction
After 24 h
3’ end formation in mammalian cells
Cleavage and
polyadenylation site
5’
AAUAAA
GU rich
3’
15-20 nt
5’
AAUAAA
OH
P
GU rich
degradation
5’
AAUAAA
A250
3’
SPECIFIC COMPLEXES ARE INVOLVED IN mRNA 3’END
PROCESSING
Cleavage and
Polyadenylation
Specific Factor
sito di taglio
CPSF
5’
AAUAAA
CA
CFII
Cleavage Stimulation Factor
CstF
3
’
PAP
GU-rich
CFI
Poly-A polymerase
Curr Opin Cell Biol. 2004 Jun;16(3):272-8
band shift/EMSA assay: to study RNAprotein interaction in vitro
Labelled RNA
In vitro transcription
1
Labelled RNA
2
Purified protein
band shift/EMSA assay: to study RNAprotein interaction in vitro
2
1
Labelled RNA
Purified protein
RNA-protein
complex
Free RNA
SPECIFIC COMPLEXES ARE INVOLVED IN mRNA
3’END PROCESSING
Cleavage and
Polyadenylation
Specific Factor
sito di taglio
CPSF
5’
AAUAAA
CFII
Cleavage Stimulation Factor
CstF
3
’
PAP
EMSA assay for
testing the
interaction
between CPSF 160
and consensus
sequence AAUAAA
CA
GU-rich
CFI
AAUAAA
Poly-A polymerase
AAGAAA
HeLa NE
Curr Opin Cell Biol. 2004 Jun;16(3):272-8
complex
sito di taglio
free RNA
3’-End Formation: RNA Processing
Cleavage and
Polyadenylation
Specific Factor
Cleavage Factors
Cleavage
Stimulation
Factor
25
26
degradation
27
28
29
CAP and polyA tail influence efficient translation
3’ end formation occurs during transcritpion
Polyadenylation is linked to termination
Torpedo Model
mRNA
AAAAAAAAA
pre-mRNA
CPSF pA
Xrn2
CstF
RNAPII
RNAPII
Polyadenylation is linked to termination
Torpedo model
Allosteric model
Variations at the transcriptome 3′ end—when processing gets alternative
Approximately up to 70 % of the transcriptome is affected by a mechanism widely
referred to as “alternative 3′ end cleavage and polyadenylation” (APA)
APA can be regulated on the level of mRNA 3′ end processing by various cis- and
trans-acting determinants:
(1)the intrinsic strength of sequence elements,
(2) the concentration or activity of polyadenylation factors,
and/or
(3) tissue- or stage-specific regulatory factors are the most important key players
APA is highly regulated during development
Proliferating cells tend to use upstream (“proximal”) PASs and produce mRNAs
with shorter 3′UTRs, while quiescent/differentiated cells favor downstream
(“distal”) PASs and produce mRNAs with longer 3′UTRs
In contrast during somatic reprogramming or tumorigenesis, proximal PASs are
favored leading to shorter 3′UTRs
APA transcript isoforms of the same gene can encode different proteins and/or
change the 3′UTR properties, leading to:
- the inclusion or exclusion of mRNA stabilizing or destabilizing elements,
- miRNA target sites,
- result in different translation efficiencies
- result in different subcellular localization
Alternative polyadenylation of the immunoglobulin μ heavy chain gene
sub obtimal pA
Secreted
terminus
Plasma cells
obtimal pA
Membrane bound
terminus
B cells
Secreted
terminus
Membrane bound
terminus
CSTF-64 levels control the alternative processing of mRNA.
In not activated B cells the limiting
concentration of CSTF allows the
recognition
of
the
stronger
polyadenylation
signal.
The
immuglobulin produced will then
contain a portion for binding to the
membrane.
After the activation of B cells CSTF
levels are increased and this allows
the
use
of
the
weaker
polyadenylation site that will be
preferentially used because it will
be the first to be transcribed.
CstF-64
The polyadenylation factor CstF-64 regulates alternative processing of IgM heavy chain premRNA during B cell differentiation.
Takagaki Y, Seipelt RL, Peterson ML, Manley JL.
Cell 1996 Nov 29;87(5):941-52
U1A: an example of on-off regulation of the cleavage and
polyadenylation step in RNA processing
U1A is a protein that binds to U1snRNA
Obtimal U1A binding site
U1A is able to bind to its mRNA
The mRNA encoding for U1A protein has a
suboptimal polyA site (AUUAAA) and 2
sequences similar to the U1A binding site
on U1snRNA
U1A
EMSA assay with increasing amount of U1A
protein revealed that U1A binds its own
mRNA and that this latter possessed two
U1A binding sites
RNA
free
-
U1A autoregulation
When the level of U1A exceeds that of U1 snRNA, the excess U1A protein binds to its
two binding sites in U1A pre-mRNAs. Since U1A binding does not prevent cleavage of
the pre-mRNAs, free 3′ ends are generated in the normal fashion
In the absence of polyadenylation, however, both cleavage products are rapidly
degraded by an exonuclease, so no functional U1A mRNA is produced. As a result,
synthesis of U1A protein is decreased until all the excess is used in formation of new U1
snRNPs.
Sub-obtimal U1A binding site
sub-obtimal site
influenza A by its connection to the cellular 3′ end apparatus has devised
an efficient way to specifically shut off cellular gene expression
The influenza A NS1 protein is one of the most abundant proteins synthesized in infected cells
It interacts with the cellular 30 kDa subunit of CPSF. By sequestering CPSF30 it prevents the
binding of the CPSF complex to the RNA substrate thus inhibits 3′ end cleavage and
polyadenylation of the host pre-mRNAs by preventing
NS1 also targets PABPN1, which inhibits the processive synthesis of long poly(A) tails catalyzed
by PAP
As mRNA processing represents a prerequisite for cytoplasmic export, the uncleaved host premRNAs are retained in the nucleus, while viral RNAs are still exported.
APA in human disease
- Loss‐of‐function mutations of globin mRNA 3′ end processing are a well‐recognized cause of
thalassemias. Thalassemia patients have different mutations that result in an alteration of the
AAUAAA hexanucleotide of the α‐globin and β‐globin genes, and invariably inactivate or
severely inhibit gene expression.
- Gain‐of‐function mutation stimulating 3′ end processing in the prothrombin (coagulation factor
II; F2) gene. The G → A mutation converts the physiologically inefficient GC F2 cleavage site
into the mechanistically most efficient CA dinucleotide, which increases cleavage site
recognition and results in an enhancement of prothrombin mRNA and protein expression. The
result in an increased risk to develop thrombosis (referred to as thrombophilia).
- defective PAP causes cell‐cycle arrest in the G0–G1 phases, the activity of PAP thus likely
reflects the proliferative activity of cells
 PAP mRNA is overexpressed in human carcinomas of the breast, colon, ovary and
pancreas and polyadenylation activity is significantly enhanced in aggressive acute leukemias
and Burkitt lymphoma compared with less aggressive chronic leukemias and normal
lymphocytes
- mRNAs with shorter 3′UTRs tend to be more stable or globally elevated eventually leading to
higher protein output
switching to shorter 3′UTRs may allow proto-oncogenes to escape from inhibition by
miRNAs, thereby resulting in oncogene activation also in the absence of genetic alterations.
Histone mRNA processing is
polyadenylation independent
Common factors in RNA processing
pathways
CPSF-73 endonucleolytic activity
Poly(A)+ mRNAs
Histone mRNAs
CPSF-73 also displays 5′ → 3′ exoribonucleolytic activity that is
responsible for the decay of the downstream cleavage product