geetika- rna editing

8/8/2019 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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