Download - Geetika- Rna Editing
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By Geetika Thakur
Ph.D I year
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Any site specific alteration in an RNA sequence thatcould have copied from template excludingchanges due to processes such as RNA splicing andpoly adenylation
(Benne and colleagues in mid 1980s)
Can affect the mRNAs, tRNAs and rRNAs
Include both the insertion and deletion of
nucleotides and conversions of one base to another
Time of Occurrence : after the gene has beentranscribed but before it is translated into proteins
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Insertion/DeletionEditing
UridineInsertion/Deletion
Baseconversion/Substitution Editing
C U A I
U C
Base substitutions most often lead to changes atthe amino acid level, whereas the insertion anddeletion of nucleotides result in frameshifts inmRNAs
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First reported in mitochondria ofkinetioplastid protozoan so termed as KRNAediting
It inserts and deletes uridylates(Us) withinmitochondrially encoded pre mRNA posttranscriptionally
Editing process requires
Guide RNAs (g RNAs)
Endoribonulcease
TUTase
RNA ligase
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It has three functional regions
I. 5Anchoring region: 4-14nut , complementaryto pre mRNA hence forms duplex with pre
mRNA immediately 3of region to be edited
II. Central Guiding Section:55-70 nut,complementary to edited sequence hencespecify the edited sequence
III. 3 oligo U tail: 5-24 nut, non encoded, addedby mt terminal uridyltransferase (TUTase),interact with purine rich regions 5 to ES, actas repository for inserted and deleted Us
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Endoribonulease DNA Ligase
Remove Us duringediting
Specific for Us
Ligate 2 moleculeswhere 5 fragmenthas a overhang orgap
3 terminaluridylyl transferase (Tutase)
Required to create the non encoded
3 oligo [U] tails of the guide RNAs (gRNAs)
Catalyses U addition/ removal tomRNA , Specific for Us
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Pre edited mRNA and gRNA associate witheach other - Anchor duplex
Endoribonuclease cleaves the pre-mRNA atmismatch, leaving a 5 phosphate and 3-cleavage product
Cleavage directed by the gRNA interactionwith mRNA .
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Results in alterations in the codingpotential or structural properties of RNA sas a result of changes in base pairing
properties of modified ribonulcleosides
C U (Deamination)
A I (Deamination) U C (Amination)
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First observed in vertebrates for mRNAencoding apoliporotein B(apo B) in whichglutamine codon(CAA) is changed to a stopcodon (UAA)
(Cheng et al 1987) Involves hydrolytic deamination at the C4
position of cytidine
N
N
= O
NH2
N
N
= O
O
Cytosine Uracil
H20
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Editosomes are a multiprotein complexcontains
cytidine deaminase activity
competence factor: act as an adaptorprotein by binding both the deaminase andthe RNA substrate
Editing process requires
Editosome Tripartite regulatory sequence
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Mooring sequence11nt motif(UGAUCAGUAUA),3 of the edited C, is necessary for editing
Spacer- 4-6 nt between mooring sequence andedited C
Enhancer/regulator- immediately upstream from
the edited C
increaseefficiency ofediting
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TRIPARTITE REGULATORY SEQUENCE
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Apolipoprotein B (apoB) gene encodes twodifferent proteins with different propertiesfrom single transcript
I. APOB-100: 100 kDa, syn in liver and secretedinto the blood plasma, function is transport ofcholesterol from blood
II. APOB-48: 48 kDa, syn in intestine, involvedin absorption of lipids in intestine
Editing is C6666 U6666 in exon 26 of the14 Kb mRNA
This creates a Stop codon, producing atruncated form of the protein (APOB48)
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Editososme holoenzyme consist of Apobec-1 &ACF
Apobec-1(apoB mRNA editing enzyme catalyticsubunit) : dimer,54kDa RNA specific cytidine
deaminase, has weak non specific RNA bindingactivity
ACF (apobec-1 complementation factor)/ASP(apobec-1 stimulating protein) : 65kDa, RNA
binding protein, binds to mooring sequence &docks apobec-1 to selectively deaminate theupstream C moiety
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EDI ME - APOLIPOPROTEI B
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Apo B function is to export lipid
Lipoproteinassembly
LDL receptorbinding (enters
cells)
Lipoproteinassembly only (excreted)
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First reported in 1988,the most prevalent typeof RNA editing
Mediated by adenosine deaminase acting on
RNA (ADAR) enzymes, converts adenosines toinosines (AI editing)
ADAR recognises partially base paired dsRNAstructure formed by intramolecular basepairing b/w editing seq & exonic/intronicediting sites sequence(ECS)
I is read by ribosomes as G causing a codon
change and potentially protein recoding
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Conversion of Ato I thru ADAR
Watson Crick
Pairing
Altered pairing
pattern
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Has three distinct domains
ds binding domain(ds RBD): Number varies
C terminal deaminase domain- catalytic
activity of enzyme Z-DNA-binding domains: function unknown
ADAR bind to RNA in a dimeric form, allowcorrect alignment of active site for its access
to target A No. of ADARs discovered: ADAR 1,ADAR 2,
ADAR 3
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Exonic region
Potential forA-to-I editing
Editing sitecomplementarysequence (ECS)
Intronicregion
Exonic regions of a messenger RNA can be editedcotranscriptionally because they often base pair withdownstream intronic regions before splicing takes place.
This forms the double-stranded structure required forediting by ADAR
RNA PolymeraseTranscriptionProcess
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Mostly the editing sites found in non coding
regions (introns & non coding RNAs) E.g - Alu repeats: fold back into hairpin
structure which act as ds substrate forADAR
A to I editing example includes Glu R Glutamine (Q)/ Arginine (R) site by ADAR 2
GLU Codon Replaced by Arginine
Decreased Ca permeability ofion channel
A to I
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Common in monocots,dicots and lower plants
chloroplast and mitochondrial transcripts
Mechanism underlying it is still unclear but
thought to be mediated by:
CTP synthetase
A pyridoxal depentent enzyme : plays role innucleotide metabolism transfers amino groupfrom glutamine to UTP for synthesis of CTP
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Base insertion /deletion and base substituion
leads to:
Creation of new start and stop codons
Removal of stop codons
Aminoacid substitutions
Alterations in splice sites
Alterations within untranslated regions (UTRs)-affect mRNA stability, translatability, andprocessing
Creation of open reading frames (ORFs)
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Editing creates essential structural elements atthe primary, secondary, and tertiary levels,involving both loop nucleotides and base-paired stems
Change tRNA identity (i.e. alter recognition byaminoacyl synthases)
Affect 5 andor 3processing
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Editing of rRNA appears to be less frequentthan alterations in mRNAs and tRNAs, butwhere it occurs, it appears to be functionallyimportant
For e.g. The single C-to-U change observed inthe small subunit rRNA in Dictyosteliummitochondria falls within the highly conserved530 loop, which is thought to play a critical
role in tRNA selection and proofreading
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Often the genomic information encoding anORF or a tRNA is cryptic or incomplete and willnot yield a functional product and isdependent on RNA editing for its biologicaloptimization and eventual survival
RNA editing could be the potential ofsynthesizing two or more distinct productsfrom a single gene as exemplified by the viral
editing systems or in the tissue-specific apoBediting
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Source of genetic variation leading to
evolution
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