Regulation of RNA Processing and RNA Editing

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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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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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.

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Regulation of RNA Processing and RNA Editing