Download Post-transcriptional modifications Cap a

Post-transcriptional modifications
5’ capping
Cap a methylated guanosine bound through a 5’-5’
triphosphate linkage to the 5’phosphate of RNA
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Steps:
1. RNA triphosphatase,
removes terminal
phosphate
2. Guanylyl transferase
adds the capping GMP
from GTP
3. Two methyl
transferases methylate
the N7 of the capping
guanosine and the 2’Omethyl group of the
penultimate nt.
Occurs early in
transcription process,
before chain reaches
30nt.
Functions:
Protection:
a cap is joined through special linkage found nowhere else
in RNA. Might be expected to protect from RNases that
start at 5’ end of substrates
Translatability
Eukaryotic mRNA gains access to the ribosome for
translation via cap binding protein.
If there is no cap, the CBP can not bind – translation is very
poor.
Transport of mRNA out of the nucleus
Proper splicing of mRNA – removal of the first intron is
dependent on the cap in vitro.
This effects may be mediated by cap-binding complex that
is involved in spliceosome formation
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Experiment: LUC mRNA with and w/o cap, with and w/o
polyA introduced in tobacco cells
Outcome: polyA and cap act synergistically to stabilize and
enhance translation of mRNA
Polyadenylation
•rRNA and tRNA do not have poly A tail.
•Process of adding poly A nts is called polyadenylation
•Most eukaryotic mRNAs and their precursors have a chain
of about 250nt long at their 3’ end.
•It is added post-transcriptionally by poly (A) polymerase.
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Functions:
Protection
Translatability
Stability
Functions:
Effects of polyadenylation on
translatability and stability of
mRNA
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Mechanism of polyadenylation:
•Transcription of genes goes beyond the polyadenylation site.
•Then transcript is cleaved and polyadenylated at the 3’end
created by cleavage.
•Polyadenylation signals: in mammalian genes – AATAAA
about 20-30 bp before the polyadenylation site.
•In RNA – sequence AAUAAA – in most mammalian RNAs
20nt before their polyA
Experiments: importance of AAUAAA was tested
First the nts were deleted between this sequence and polyA –
deletion shifts the polyA site downstream ~ the number of nts
deleted
•No deviation in AATAAA could occur w/o destroying
polyadenylation
•AAUAAA is not sufficient for polyadenylation, otherwise
polyadenylation would occur downstream of AAUAAA
sequences in many introns, but it does not
•Polyadenylation is destroyed by deleting sequences
immediately downstream of polyadenylation site. Region
downstreams might contain another element of
polyadenylation signal. This signal is not highly conserved,
but is GT or T reach.
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•Thus: efficient mammalian polyadenylation signal
consists of an AAUAAA motif about 20nts upstream of
polyadenylation site in the re-mRNA, followed 23 or 24
nt later by a GU-rich motif, followed immediately by the
U-motif.
•Plant signals usually contain the AAUAAA motif, but
more variation in this region is allowed that in
mammalian AAUAAA.
•Yeast polyadenylation signals are more different, and
rarely contain the AAUAAA.
Polyadenylation requires cleavage of the pre-mRNA
and polyadenylation at the cleavage site.
Cleavage – proteins –
CPSF – cleavage and polyadenylation specificity
factor
•CstF - cleavage stimulation factor
•CFI – cleavage factor I
•CFII – cleavage factor II
•polyA polymerase
•And RNA polymerase II, in particular the CTD of
RPB1.
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Initiation of polyadenylation
•The signal to initiate the polyadenylation of the cleaved
substrate – AAUAAA followed by at least 8 nt.
•Once polyA reaches about 10nt, further polyadenylation
becomes independent of the signal and depends on polyA
itself
•Two proteins participate in initiation of the process: CPSF
that binds to the AAUAAA motif and polyA polymerase
Elongation
•Required specificity factor called PolyA binding protein
(PABII). Binds to a preinitiated oligoA and aids polymerase
to elongate the polyA to 250nt
•PABII acts independently of the AAUAAA motif
•It depends only of polyA and its activity is enhanced by
CPSF.
Gene silencing
Multiple copies of transgenes introduced into plant cells can
interact with each other and with homologous host genes in
trans, resulting in the inactivation of expression of both
genes.
This phenomenon called homology-dependent gene
silencing (HDGS) occurs generally in plants and is similar to
certain types of gene silencing described for fungi, protozoa,
nematodes, insects and mammals.
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Gene silencing
The results of nuclear run-on transcriptioin experiments
suggest there are:
Transcriptional (TGS) and
Posttranscriptional (PTGS) forms of HDGS.
Both types of HDGS are often associated with de novo DNA
methylation, which is usually confined to promoters in cases
of TGS and transcribed regions in instances of PTGS.
PTGS
In the case of PTGS, the interaction in trans of genes with
similar transcribed sequences results in sequence-specific
degradation of RNAs derived from the genes involved.
Highly expressed single-copy loci, transcribed inverted
repeats, and poorly-transcribed complex loci can act as
sources of diffusible signals that trigger PTGS.
In plants, posttranscriptional gene silencing (PTGS) is
implicated as an antiviral mechanism.
Intercellular spreading of the PTGS effect allows the plant to
respond in a systemic manner after a localized viral
challenge.
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Gene silencing
In some cases, mobile, sequence-specific silencing signals can
move from cell-to-cell or even over long distances in the plant.
Several current models hold that silencing signals are “aberrant”
RNAs (aRNA), that differ in some way from normal mRNAs.
The most likely candidates are small antisense RNAs (asRNA)
and double-stranded RNAs (dsRNA).
Most current models assume that silencing signals interact with
target RNAs in a sequence specific fashion.
This results in degradation usually in cytoplasm, by exonucleolytic
as well as endonulceolytic pathways, which are not necessarily
PTGS specific.
Possible mechanisms
Gene silencing is probably often the result of more than one
mechanism.
Transcriptional gene silencing (TGS) is often associated with
methylation of the gene, which may inhibit transcription.
In posttranscriptional gene silencing (PTGS), high levels of
normal mRNA can cause activation of RNA-dependent RNA
polymerases (RdRP), which can synthesize antisense transcripts.
Antisense transcripts can also be synthesized when a gene is
present in high copy number, especially where tandem-inverted
repeated copies are present.
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Possible mechanisms
Double-stranded RNAs resulting from either RdRP activity or
base-pairing between antisense transcripts and mRNAs become
targets for ribonucleases, which degrade dsRNAs into small
fragments of about 21-25bp.
This process appears to be part of the natural defense against
viral dsRNAs.
Small dsRNAs may serve to target nuclear copies of the gene for
methylation, resulting in a feedback mechanism for gene
silencing.
dsRNAs can also be transmitted intercellularly via
plasmodesmata, causing systemic gene silencing.
Again, this systemic component of gene silencing is likely part of
the natural defense against viruses.
In RNAi, introduced double-stranded RNA (dsRNA) (i) is
recognized by proteins (ii) which, starting from the dsRNA termini,
cleave it (iii) into ~22 nt fragments to produce nucleoprotein
complexes (iv).
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The strands of the short dsRNA are separated and one strand is
used as a guide to recognize single-stranded RNA (ssRNA) with
complementary sequences (v). Each complex cleaves the ssRNA
at a position approximately in the middle of the guide sequence (v,
vi).
PTGS in
plants
In PTGS in plants (b), an RNAi-like cleavage mechanism
operates. The dsRNA directing the mechanism is introduced
by a replicating virus or an inverted repeat transgene that
produces self-complementary (hairpin) RNA.
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PTGS in
plants
The short RNAs from the cleavage (or alternatively uncleaved dsRNA) enter the nucleus to guide a
methyltransferase complex to sequences for methylation and
also spread into other cells to direct the cleavage of
homologous ssRNAs.
PTGS in
plants
The possible points of action of viral silencing suppressors,
p25 and HC-Pro, are shown. p25 prevents the spread of the
mobile PTGS signal but does not inhibit existing PTGS. HCPro is a viral silencing suppressor.
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Posttranscriptional RNA
modification -editing
RNA editing – is a posttranscriptional changes of RNA
sequences involving:
a) Insertions
b) Deletions
c) Substitutions (conversions)
…of nucleotides within an RNA chain.
In contrast:
Capping, splicing, 3’ end formation, trimming of ends,
modification of bases (e.g. methylation), etc., etc.
… are RNA processing.
RNA editing
Type of RNA editing Base Mechanism
Posttransciptional
insertion and
deletion of
nucleotides.
U
Guide RNAs
C
?
Cotranscriptional
insertion of
nucleotides
A
Modificationsubstitution of
nucleotides
C
(to U)
Polyadenylati
on
Polymerase
stuttering
Deamination
G
U
(to C)
A
(to I)
Genes and Organism
Mitochondrial genes of
Trypanosomes
Mitochondrial genes of
Physarum polycephalum
Mitochondrial genes of
vertebrates
Phosphoprotein gene of
Paramyxoviruses
Apolipoprotein gene of
mammals.
Mitochondrial and chloroplast
genes of plants
Wilm’s tumor susceptibility
gene, WT1 of mammals
Mammalian glutamate receptor
genes, matrix gene of
Measles virus.
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Conversions
Insertion/deletions
Examples
1. Transcipts of
trypanosomes
2. Mitochondria of P.
polycephalum
3. Paramyxoviruses (P
gene)
4. Mitochondria of
vertebrates
5. Apolipoprotein B gene
of mammals
6. Mitochondria of higher
plants
7. Chloroplasts of higher
plants
8. Glutamate-gated ion
channels of mammals
..AAUUUAUGUUGUCUUU
..AUCUCUAAGGGUUUAACCGG
Frameshift
..AUUAAAAAGGGGCACAC…
Frameshift
..CAGUAAAAAAAAAA…
Stop C
..GAUAUAAUUUGAUCAGUAUA…
C
Stop
C
..UUUUUCAUUGUGGUUUAC…
PHE
TYR
C
..AAUAAUAUGGCGAAACAU…
Start
A
..UUUAUGCGGCAAGGA…
ARG
Discovery of RNA Editing
It was noted early on that several of the structural genes had
frameshifts and lacked translation initiation codons.
In 1986, Benne et al. discovered that U residues were inserted
into the mRNA after transcription and that this overcame the
frameshifts.
U deletions also were found to occur at a lower frequency.
The extent of this "RNA editing" in the lizard Leishmania, L.
tarentolae, is indicated.
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Discovery of RNA Editing
Several mRNAs are edited at the 5' end and several are" panedited" throughout the gene, creating an open reading frame from
nonsense sequence.
Discovery of RNA Editing
Blum et al. discovered that a novel class of small RNA molecules,
transcribed both from the maxicircle and the thousands of
minicircles, contained the editing information.
The gRNAs were discovered by a computer search for short
maxicircle sequences complementary to edited mRNA
sequences.
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Discovery of RNA Editing
These guide RNAs or gRNAs had a 5' sequence complementary
to the mRNA just 3' of the first editing site.
They also had a non-encoded 3' oligo[U] tail.
The central portion of the gRNA contained sequence that was
complementary to edited mRNA sequence, provided G was
allowed to pair with U and well as with C.
Therefore the gRNA would form a perfect duplex with edited
mRNA except for the oligo[U] tail.
Reading:
p. 471-488, 521-526
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