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8/12/2019 Regulation of RNA Processing and RNA Editing
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RNA PROCESSING
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RNA capping the first modification of pre-
mRNA
RNA pol II produce 25 nts of RNA the 5 end of thenew RNA molecule is modified by addition of the cap7 methyl guanosine.
Three enzymes acting are1. Phosphatase- removes phosphate from 5 end of nascent
RNA.2. Guanyl transferase- adds GMP in the reverse linkage (5 to 5)
3. Methyl transferase- adds methyl group to guanosine
5 cap
It distinguishes other RNA present in the cell. Cap binds a protein complex called CBC (cap-binding
complex). It helps the RNA to properly processed andexported.
It has important role in translation of mRNAs in the
cytosol
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RNA pol II pauses and the
kinase positive transcriptionelongation factor b (P-TEFb)phosphorylates RNA pol II onthe serine 2 residue in therepeat unit to C-terminal
domain (CTD) of the largesubunit of the enzyme.
Synthesis of the cap iscarried out by the enzymestethered to the CTD of pol II.
The cap remains tethered tothe CTD through anassociation with the cap-binding complex (CBC)
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P-TEFb is composed ofcyclin dependent kinase
(CDK9) and either cyclin T1,
T2 or K.
This terminal is also called C-terminal domain kinase1
(CTDK1).
The pausing and regulatory
phosphorylation event allowsfor the potential of
attenuation in the rate of
transcription.
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5 Cap
Type 1 cap: the ribose
(O2) gets methylated (and
the first base if A-N6 gets
methylated).Type II cap: in some
species the subsequent
residue at +2 position is
also methylated again atO2of ribose
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The capping reaction can be used to regulate theprotein synthesis, a strategy utilized by some
animals during egg maturation.
Most RNA viruses cap their genomes and mRNAs
whilst the Picornaviridae, whose infection strategyexploits their lack of cap dependence, block the 5
end of their genome with a viral protein.
The orthomyxoviridaedo not cap their genome
segments but steal pre-formed caps from the hostmRNAs, a transesterification process which has
been termed capsnatching
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Polyadenylation of mRNA
A specific sequence is recognized by the CPSFendonuclease activity of the polyadenylate
polymerase (PAP) which cleaves the primary
transcript at 3 end of the mRNA.
Initial polyadenylation is slow because PAPdissociates after adding each adenylate residue.
After synthesis of short stretch the PABP attaches to
the tail and increases the processivity
A stretch of 20-250 A residues is then added to the3end by the polyadenylate polymerase activity.
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The mRNA site wherecleavage occurs ismarked by the twosequence elements
The conserved
sequence 5AAUAAA3upstream 10-30 nts on5 site(cleavage site).
Sequence rich in G
and U residues, 20-40nts downstream of thecleavage site.
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i. CPSF (cleavage andpolyadenylation specificityfactor).
ii.CstF (cleavagestimulation factor).
iii.Two cleavage factorproteins (CFI and CFII).
After cleavage, the enzymepoly(A) polymerase (PAP)adds A nucleotides to the 3end of the RNA, using ATPas a substrate. PAP is
bound to CPSF during thisprocess.PABII (poly(A) bindingprotein II) binds the poly(A)tail as it is produced
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Histone mRNAs and the genomes of certain plantviruses are not polyadenylated.
A secondary structure adopted by the histone
transcripts is responsible for the 3 end maturation,
which involves U7 snRNA and associated proteins The poly (A) tail and its associated proteins probably
help protect mRNA from enzymatic destruction.
Many bacterial mRNA also acquire poly(A) tails, but
these tails stimulate decay of mRNA rather thanprotecting it from degradation.
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INTRON
Initially described inAdenovirus and later inthe ovalbumin gene ofthe chicken The isolated ovalbumin
gene was denatured andrehybridized with mRNAfrom a chicken egg
The hybrids were
examined using electronmicroscopy
D loops formed,representing singlestranded regions of
genomic DNA not present
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Self splicing introns
First releaved in 1982 in the studies of splicingmechanism of the group I rRNA intron from the
ciliated protozoan Tetrahymena thermophila,
conducted by Thomas Chech and colleagues.
They transcribed isolated tetrahymena DNAincluding intron in vitrousing purified bacterial RNA
polymerase.
The resulting RNA spliced itself accurately without
any protein
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Self- splicing intron
The 2 most common self-splicing mechanism are1. group I intron
2. group II intron
Group I intron are found in nuclear, mitochondrial
and chloroplast genes. Group II in mitochondrial and chloroplast mRNA in
fungi, algae and plants primary transcript.
Many of the group I and group II are self splicing and
do not require ATP hydrolysis.
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Group I
It requires externalguanosine nucleotide as a
cofactor.
The 3-OH of the
guanosine nucleotide actsas a nucleophile to attack
the 5 phosphate of the 5
nucleotide of the intron and
covalently attaching thetwo exons together.
The spliced intron is
eventually degraded.
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Group II
Group II intron are
spliced (withoutexternal nucleophile)the 2 OH of Adenineresidue within theintron.
This residue attack the3 nucleotide of the 5exon forming aninternal loop called alariat structure.
The 3 end of the 5exon then attacks the5 end of the 3 exon asin group I splicingreleasing the intron andcovalently attaching thetwo exons together.
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Splicing by spliceosome (Group III)
Here the splicing is catalysed by specialised RNA-protein complexes called small nuclear
ribonucleoprotein particles (snRNPs)
The RNAs found in snRNPs are identified as
U1(165bs), U2(188bs), U4(142bs), U5(116bs) andU6(107bs).
The genes encoding these snRNAs are highly
conserved in vertebrate and insects and are also
found in yeasts and slime moulds indicating theirimportance.
Spliceosome introns generally have the dinucleotide
sequence GU at 5 end and AG at 3end.
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The U1 RNA has sequences that are complementaryto sequences near the 5 end of the intron.
The binding of U1 RNA distinguishes the GU at 5
end of the intron from other randomly placed GU
sequences in mRNAs. The U2 RNA also recognizes sequences in the
intron, in this case near the 3 end (branch point).
The addition of U4, U5 and U6 RNAs forms a
complex spliceosome. This then removes the intron and joins the two exons
together.
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The U1 snRNP forms base
pairs with the 5 splice
junction, and the BBP and
U2AF recognise the branch
point
The U2 snRNPdisplaces BBP and
U2AF and forms base
pairs with the branch
point site consensus
sequence.
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Subsequent
rearrangements create
the active site of the
spliceosome and position
the appropriate portions
of the pre-mRNA
substrate for the first
phosphoryl-transferasereaction.
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Several more RNA-RNArearrangements occur that
break apart the U4/U6
base pairs and allow the
U6 snRNP to displace U1at the 5 splice junction to
form the active site for the
second phosphoryl
transferase reaction, which
completes the splice.
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SPLICEOSOME USES ATP HYDROLYSIS
The exchange of U1 snRNP for U6snRNP occursbefore the first phosphoryl-transfer reaction.
This exchange requires the 5 splice site to be read
by two different snRNPs thereby increasing the
accuracy of 5 splice site selection by thespliceosome.
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SPLICEOSOME USES ATP HYDROLYSIS
The branch point is first recognised by BBP andsubsequently by snRNP, this check and recheck
strategy provides increased accuracy of site
selection.
The binding of U2 to the branch point faces theappropriate adenine to be unpaired and thereby
activates it for the attack on the 5splice site.
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SPLICEOSOME USES ATP HYDROLYSIS
After the first phosphoryl-transfer reaction has occured a
series of rearrangement brings the two exons into closeproximity for the second phosphoryl transfer reaction.
The snRNAs both position the reactants and provide thecatalytic sites for the two reactions.
The U5 snRNP is present in the spliceosome before this
rearrangement occurs.
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Regulatory elements in pre mRNA Cis-regulatory elements in pre-mRNA splicing.
Information in pre-mRNA substrate contributing to the splicesite recognition includes short and consensus sequences at 5splice site(ss), 3 branch point site(BPS) which are typicallylocated 30-50 nts upstream of the 3 ss in human.
Polypyrimidine tract (PPT) is just downstream of the BPS. But
this is short sequence and relatively degenerative. Additional flanking ciselements in pre-mRNA are required to
facilitate splice site recognition and selection.
Based on the position and function these ciselements aredivided into 4 categories.
ESEs, ESSs, ISEs, ISSs.
They serve as binding sites for trans regulatory factors, suchas members of SR and hnRNP protein families, which in turnregulate splicing by either promoting or preventing therecruitment of basal splicing machinery.
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Alternative splicing are regulated Splicing factors bind to exonic (or intronic) splicing enhancers (ESE or ISE) or silencers (ESS and
ISS) to regulate splicing.
1. Splicing enhancers are recognized by SR proteins.
2. Splicing silencers are recognized by hnRNPs: (it lack SR domain)
The ultimate alternative splicing decisions are therefore made by combinational effects of the similar(synergic) or opposing (antagonistic) splicing regulatory signals.
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For short introns (250), spliceosomal componentsfirst assemble across and exon, a process called exondefinition.
Exon definition:- U1 snRNP binds to the 5 ss downstream ofthe exon, components of U2 snRNP associate with thepolypyrimidine tract/3 ss and BPS upstream of the exon,respectively.
Regulatory sequences within the exon recruit protein factorssuch as the SR protein family members, which bridge andcross exon interaction and stabilize the exon definitioncomplex.
Since catalytic steps of the splicing take place across anintron, the cross exon complex must be switched to cross
intron complex, a process that is currently not well
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ESE - exon splicing enhancer sequences
SRESE binding proteins
Pre mRNA splicing by major spliceosome
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Accuracy of splice-site selection
1. Co-transcriptional loading: factors bound to the 5 siteare poised to interact with the factors binding to the
next 3 site
2. SR (serine argininerich)proteins bind to Exonic
Splicing Enhancers and recruit the splicing machinery.They ensure that splice sites close to exons are
recognized preferentially.
SR proteins not only ensure the accuracy and
efficiency of constitutive splicing, but also regulate
alternative splicing.
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Coordination ofsplicing and
transcription
provides anattractive
mechanism for
bringing the twosplice sites
together
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SPLICING
ERRORS
Exon skipping: very fast
transcription rate and/orinefficient tetheringcould cause exonskipping.
Cryptic splice site:
cryptic splicing signalsare nucleotidesequences of RNA thatclosely resemble truesplicing signals
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Alternate splicing
Alternative slicing is an important layer of geneexpression control and enhances the proteomicdiversity.
Recent studies show that more than 94% humansgenes undergo AS events (Pan et al.2008; Wang et
al.2008) mRNA transcripts produce only one mature mRNA
and one corresponding polypeptide.
Others can processed in more than one way to
produce different mRNAs and thus differentpolypeptide.
AS regulations have been demonstrated to playpivotal roles in different cell types, developmental
stages, across tissues, sex determination and inres onse to external stimuli.
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I. Alternative splicingPoly (A) site choice
If there are more than
one cleavage site and
polyadenylation,
This use of one closest
to the 5 end will removemore of the primary
transcript sequence.
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II. Alternative splicing
Alternative splicing
patterns produce form
a common primary
transcript
3 different forms ofthe myosin heavy
chain at different
stages of fruit fly
development.
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Both mechanism I and II
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The primary transcript has the molecular signal for
alternate splicing and this pathway is determined by
the processing factors, RNA binding proteins that
promote one particular path.
The basic AS patterns can be classified into1. Cassette exon inclusion or skipping.2. Alternative 5 splice site
3. Alternative 3 splice site
4. Intron retention
5. Mutually excusion alternative exons6. Alternative promoter and first exon
7. Alternative poly A site and last exon
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Additionalpatterns of
alternative
splicing
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AS is generally controlled by cooperative interplays
between RNA binding proteins (RBPs), regulatory
elements in nascent transcripts and the basal
splicing apparatus.
Splicing regulating RBPs bind directly to splice sites,or interact with specific sequences in pre-mRNA to
facilitate or block the recruitment of splicing
machinery, which in turn stimulate or repress splice
site usage(modulating alternative splicing)
AS h i t diff t t f th
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AS choices occur at different stages of the
splicing process.
Splice site recognition:-understood by splicingregulation of survival ofmotor neuron (SMN)exon7.
Splice site pairing:-prevention of Fas (CD95)exon 6 inclusion by RNAbinding motif protein 5
(RBM5) Combinational effect of
both : polypyrimidine tractbinding protein
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Temporal control of alternate splicing
Influenza A M1
mRNA
Early:
Splicosome
recognizes M3
splicesitemakes M3
mRNA
Late: Viral P
proteins recruit
cellular SR
protein, directing
splisosome to
M3 splice site to
make M2 mRNA.
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Tissue specific splicing regulators
1. Presence of NOVA1 and NOVA2 (Ule et al.2006,2005)
2. nPTB (Boutz et al. 2007)
3. FOX1 and FOX2 (Gehman et al. 2011; yeo et al. 2009)
4. RBM20 (Guo et al.2012)
5. RBM35(Warzecha et al. 2009)
6. RBM11 (pedrotti et al. 2011)
Ubiquitously expressed splicing regulators also
participate in the tissue specific alternative splicing
regulation.
Differentially expression of tissue specific splicing
regulators and /or variable concentrations and/or
modifications of ubiquitously expressed splicing
regulators will also regulate the tissue specific
splicing regulators.
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Alternative splicing and human disease
Defects in splicing are regarded as a primary cause
of diseases and mechanisms of which can be
classified into two main groups
1. Disruption of ciselements: splice sites, splicing cis
regulatory elements.
2. Disruption of transacting factors : component of core
splicing machinery and splicing regulators.
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Cis effects: disruption of splicing code They are inherited human disorders.
These mutations cause1. Change in encoded protein
2. Alter the ratio of natural protein isoforms
3. Premature termination codon
Duchenne muscular dystrophy (DMD) mutation in massivedystrophin gene. Ex:- TA substitution in exon 31 simultaneouslycreate a premature stop codon and an exonic splicing silencer(ESS),leading to exon 31 skipping and mild form of DMD.
Frontotemporal dementia and parkinsonism linked chromosome 17(FTDP17), which is an autosomal dominant disorder caused bymutations in the MAPT gene that encodes tau. Numerous point mutations within and around MAPT exon 10 destruct the
exonic or intronic splicing elements and alter normal 1:1 ratio of isoforms
with or without exon 10. The disrupted splicing in turn destroys the balance between tau proteins
containing either four or three microtubule-binding domains (R) andcauses FTDP17.
Trans effects: defects in splicing
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Trans effects: defects in splicing
machinery/regulators
Mutation or stoichiometric changes in these regulators
can extensively change the AS patterns of their targets. Autosomal dominant form of retinitis pigmentosa (adRP)
that caused by mutations in five proteins belonging toU4/U6-U5 tri-snRNP:PPRF31, PPRF8, PPRF3, RP9 and
SNRNP200. Another example is spinal muscle atrophy (SMA) that
cause the mutations in SMN gene which plays a complexrole in snRNA biogenesis. It is found that snRNA isaffected differently in distinct tissue of SMN-deficient
mouse rather than a uniform decrease. Point mutation in U4atac snRNA (component of minor
spliceosome) linked with microcephalic osteodysplasticprimordial dwarfism type I (MOPD-I i.e.,Taybi-Lindersyndrome)
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mRNA stability
The concentration of any molecule depend on Rate of
synthesis and rate of degradation.
Change in steady state may accumulate or deplete the
mRNA.
Average half life of a vertebrate mRNA 3hrs, with the pool
of each type of mRNA turning over about 10 times per
cell generation.
Half life of bacterial mRNA is about 1.5min.
mRNA s are degraded by ribonucleases.
In E.colimany cuts mRNA by endonuclease followed by 3 to 5
degradation by exonuclease.
A hairpin structure in bacterial mRNAs with a rho-independent
(hairpin) confers stability against degradation.
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In lower eukaryotes the major pathway involves first
shortening the poly(A) tail, then decapping and
degrading the mRNA in the 5-3 direction.
Higher eukaryotes
3-5 degradation is major pathway in higher eukaryotes. All eukaryotes have conserved 10 exosome (3 5
exonuclease) involved in processing of 3 end of mRNAs,
rRNAs, tRNAs, snRNAs and snoRNAs.
Control of mRNA stability mRNA for milk casein has half life of 1 hrs. When stimulated
with prolactin half life increases to 40 hrs.
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tRNA splicing
These introns are spliced by a specific splicing
endonuclease that involves a cut and paste
mechanism.
In order for tRNA intron removal to occur the tRNA
must first be properly folded into its characteristicoverleaf shape.
Misfolded precursor tRNAs are not processed which
allows the splicing reaction to serve as a control
step in the generation of mature tRNAs.
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tRNA processing
Endonuclease P
tRNA nucleotidyltransferase.
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Modified bases in tRNA
10% of nucleotides become modified during tRNA synthesis usuallyby posttranscriptional chemical modification.
Eukaryotic rRNA Processing
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Eukaryotic rRNA Processing
The 45S precursor is methylated
at more than 100 of its 14,000
nucleotides, (bases or 2-OH)(Uridinespseudouridine)
Enzymatic cleavage of the 45S
precursor produces the 18S, 5.8S
and 28S rRNAs and assembleswith ribosomal proteins. All
processing require small
nucleolar RNAs (snoRNA) found
in protein complexes (snoRNPs)
in the nucleolus that are
reminiscent of spliceosomes.
The 5S rRNA is produced
separately
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Bacterial rRNA processing
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Special function RNAs undergo processing
snRNAs & snoRNAs are synthesised as large
precursors and then processed.
Some snoRNAs are encoded within the introns of
other genes. After splicing snoRNP binds to snoRNA
and remove extra RNA at both ends.
Pre-snRNA are synthesised by RNA pol II,
ribonuclease remove extra RNA at each end.
snRNA undergo modification.
The end