RNA Editing

Definition: any process, other than splicing, that

results in a change in the sequence of a RNA
transcript such that it differs from the sequence of
the DNA template
First considered a bizarre relic; now recognized as
widespread

RNA editing has been reported in:

protozoa, plants and mammals, not yet fungi or
prokaryotes; nuclear, mitochondrial, chloroplast, and
viral RNAs; mRNA, tRNA, rRNA
 RNA editing
Two general types
Base modification (deaminase)

A to I double-stranded mechanism, seen in viruses,

human genes

C to U, U to C seen in chloroplasts, plant mitochondria,

human genes

Insertion/deletion
U insertion/deletion, seen in kinetoplastid protozoa
mono/di nucleotide insertion, seen in Physarum
nucleotide replacement, seen in Acanthamoeba
tRNAs
Where were the real functional genes?

• Investigators generated cDNA clones to some of the

kinetoplast mRNAs and sequenced them
• Sequences were partially complementary to
pseudogenes on maxicircle DNA

cytochrome oxidase
subunit II

– the COXII DNA sequence above is missing 4 Us

found in the mRNA
• Called this “Editing” because it produced functional mRNAs and
proteins from pseudogenes
 Editing Mechanism
• Post-transcriptional
• Guide RNAs (gRNAs) direct editing
– gRNAs are small and complementary to portions
of the edited mRNA
– Base-pairing of gRNA with unedited RNA gives
mismatched regions, which are recognized by the
editing machinery
– Machinery includes an Endonuclease, a Terminal
UridylylTransferase (TUTase), and a RNA ligase
• Editing is directional, from 3’ to 5’
 Guide RNAs Direct Editing
in Trypanosomes.

Editing is from 3’ to 5’ along an unedited RNA.

RNAs THAT FUNCTION IN RNA PROCESSING
rRNA
snoRNAs form complexes with protein, direct nt modifications
snoRNAs also modify tRNAs, and likely other RNAs
tRNA
RNase P has both RNA and protein components

mRNA
snRNPs U1,2,4,5,6 form spliceosomes with many proteins
gRNAs provide sequence information for RNA editing
miRNAs important for regulating gene expression
siRNAs important for regulating gene expression
 Issue #1
• Do we need to describe multiple RNA types,
in this case:
– mRNA (with features, e.g edits)
• Pre-edited mRNA
• Intermediate, partially edited mRNAs
• Fully edited mature mRNA
– gRNA (Exists)
• Anchor site region (Exists), including G:U mismatch pair
• Editing template region
• polyU tail (post-transcriptional modification)
 Major Groups of Eukaryotes
rhizaria
alveolates
plants
chromists

Cerc
Ch
l or

omon
amoebozoa

ar
ac

ads
hn
ion

ytes
Haptophphytes
Crypto

discicristates

opisthokonts
excavates
Courtesy, S. Baldauf
Archaea
Trypanosomes

Unusual organelles:
• Flagellum
• Paraflagellar rod
• Subpellicular microtubules
• Surface membrane is densely
packed with a protein called VSG
(variant surface glycoprotein)
• Kinetoplast: Region of the
mitochondrion containing highly
packed DNA (20% of total)
• Glycosomes: peroxisomes,
contain all the glycolyitc enzymes
 The Kinetoplast
DNA with a peculiar network structure
Disk shape structure near the flagellar basal body

Lu et al. Mol. Cell. Biol.(1998)18, 2309-2323

 KDNA components
• The network contains 5-20 x103
catenated minicircles (0.8-2.5 kb) and 20-
50 catenated maxicircles (23-36 kb)

• Maxicircles: 20-40 kb. Functional

homologue of mitochondrial DNA: Region
encoding ribosomal RNAs, subunits of
respiratory complexes and a variable
region non-transcribed. Maxicircles
transcripts undergo RNA editing.

• Minicircles: 0.5-2.5 kb. Encode the guide

RNAs. All minicircles share the UMS
 RNA EDITING IN KINETOPLASTIDS

• The precursors of messenger RNAs (pre-mRNAs) have their

coding information remodeled by the site-specific insertion
and deletion of uridylate (U) residues.
•Pre-mRNAs are encoded in the maxicircle DNA
• This process creates initiation and termination codons,
corrects frameshifts and even builds entire open-reading
frames from nonsense sequences.
• The edited transcripts are translated into components of the
oxidative phosphorylation: subunits of complex I (NADH-UQ
reductase), III ( Cyt bc1), IV (cyt oxidase) and V (ATP
synthase)
• Minicircles encode guide RNAs (gRNAs) that specify the
editing.
 Pre-edited mRNAs on Maxicircle;
guide RNAs on Minicircle
ND8
ND7
COIII gRNA 1
ND9 CYB
A6
MINICIRCLE
(>1000 different molecules)
MAXICIRCLE
(~50 copies) 22kb CR3 ~1kb
gRNA 2
CR5 COII
CR4 gRNA 3
RPS12 MURF2

...UUAGGGGGAGGAGAGAuAGuuAuuAuAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG...
...AAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGUUGUGAUUUUGGAGUUAUAG...
...AAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGUUGUGuuAUUGGAGUUAUAG...
...AGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGUUGuuUGuuAUUGGAGUUAUAG...
...AAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGGuuUGuuAUUGGAGUUAUAG...
...GAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAuuuGGuuUGuuAUUGGAGUUAUAG...
...GCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG...
...AGGAAAGGUUAGGGGGAGGAGAGAAGAAAGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG...
...GAAAGGUUAGGGGGAGGAGAGAAGAAAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG...
...AAAGGUUAGGGGGAGGAGAGAAGAAuAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG...
...AGGUUAGGGGGAGGAGAGAAGAuuAuAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG...
...GUUAGGGGGAGGAGAGAAGuuAuuAuAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG...
...AAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGUUGUGAUUGGAGUUAUAG...
····|··|·|·|||·||||·||||···|||··||||·||·||·|||||·||||||||||
|·||||·||||···|||··||||·||·||·|||||·||||||||||
||·||||···|||··||||·||·||·|||||·||||||||||
||||···|||··||||·||·||·|||||·||||||||||
||···|||··||||·||·||·|||||·||||||||||
··|||··||||·||·||·|||||·||||||||||
||··||||·||·||·|||||·||||||||||
·||||·||·||·|||||·||||||||||
|·||·|||||·||||||||||
·||·|||||·||||||||||
·|||||·||||||||||
||·||||||||||
|·||||||||||
UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
UUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
UAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
UAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
UAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
UGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
CAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
UUUUAGACUAAGCAAUAGCCUCAAUAUC...
CUAAGCAAUAGCCUCAAUAUC...
UAAGCAAUAGCCUCAAUAUC...
GCAAUAGCCUCAAUAUC...
UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAA
UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAA
UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGAC
UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGA
UUUUUUUUUUUUAUUAAUAGUAUAGUGACAG
UUUUUUUUUUUUAUUAAUAGUAUAGUGA
UUUUUUUUUUUUAUUAAUAGUAUAG
UUUUUUUUUUUUAUUAAUAGUA
UUUUUUUUUUUUAUUAAUAG
UUUUUUUUUUUUAUUAA
UUUUUUUUUUUUA UAGCCUCAAUAUC...

U Tail Information Anchor

Courtesy of K. Stuart
 Issue #2

• Need multiple types of mitochondrial

genome sequence types
– Maxi-circle (Exists)
• mRNA features
– Mini-circle
• gRNA features
 The extent of editing varies between
mRNAs from the same organism

• Occurs only with mitochondrial genes encoded in maxicircles

(12)
• No editing
– 5 genes produce RNAs that do not appear to be edited: cox1,
MURF1, ND1, ND4, ND5
• Limited editing:
– 5’end of CYb, MURF2, cox2, ND7 and cox3 (L. t. and C. f.), ND8 (C.f.)
• Extensive editing (pan edited cryptogenes):
– T. brucei: ND7, cox3, MURF4, CR1-6, G1-6
– L. tarentolae; 5’portion of ATP6 RNA, entire G6 transcript, ND8,
ND9, G3, G4 and ND3
Some genes are very heavily edited!
COXIII
Cytochrome
oxidase III

From Trypanosoma
brucei

Lower case Us
were inserted
by editing.
The deleted Ts
(found in the
DNA) are
indicated in
upper case.
 Issue #3
• Do we need to indicate the type and
style of editing?
– Types include :
• U - Deletion (Exists)
• With reference to DNA would be T deletion
• U - Insertion (Exists)…would be a T insertion
– Styles include:
• Single/few edits
• 5' editing
• Pan editing
 Guide RNA (gRNA)

• Complementary to edited
RNA sections if G.U is
allowed
• Small RNAs (40-70 nt)
• Guide because they are
not conventional
templates
• Structural elements:
anchor, informational part
and Oligo(U)tail
 RNA editing mechanism
Deletion editing Insertion editing
5'- ... A UUU UG ... - 3' 5'- ... UGAU ... - 3'
3'- U AC - 5' 3'- AAUA - 5'
C
G

Cleavage
Endoribonuclease
H
UO
5'- ... A UU p UG ... - 3' 5'- ... UG OH p AU ... - 3'
3'- U AC - 5' 3'- AA UA - 5'
C
G
U addition

UMP
U removal TUTase UTP

Exo Uase
O H
5'- ... A OH p UG ... - 3' 5'- ... UG UU p AU ... - 3'
3'- U AC - 5' 3'- AA UA - 5'
C
G

Ligation
RNA ligase
5'- ... A UG ... - 3' 5'- ... UG UU AU ... - 3'
3'- U AC - 5' 3'- G C A A UA - 5'
 Editing is catalyzed by a multiprotein complex
 Editing is catalyzed by a multiprotein complexes that have not been fully defined yet. A
complex has been purified from glycerol gradients that contains the four key enzyme
activities: 20S editosome
 Endonuclease: cleavage in vitro occurs at an unpaired nucleotide immediately
upstream of the gRNA-mRNA anchor duplex.
 Exonuclease: exoUase removes non-base-paired U nucleotides after cleavage of
deletion editing sites
 TUTase:In insertion editing, Us are added to the 3’ end of the 5’ pre-mRNA
fragment by a terminal uridyl transferase as specified by the gRNA.
 RNA ligase: the natural editing ligase
substrates are nicked dsRNAs that are
completely base-paired after the correct addition
or removal of U nucleotides
 Helicase: each gRNA must be displaced from
the sequence that it creates to enable binding
by the subsequent gRNA and also from the
mRNA completely before translation
 Other 20S editosome proteins
 The Kinetoplast and Its DNA

Trypanosoma brucei

ND8
ND7
COIII gRNA 1
ND9 CYB
A6

22kb CR3
ca. 1kb
gRNA 2
CR5 COII
CR4 gRNA 3
MURF2
RPS12

MAXICIRCLE MINICIRCLE
(~50 copies) (>1000 different molecules)
Courtesy of K. Stuart
 Sequence of T. brucei ATPase Subunit 6 (A6) mRNA

AAAAAUAAGUAUUUUGAUAUUAUUAAAGUAAAuAuGuuuuuAuuuuuuuuuuGuGAuuuA
UUUUGGuuGCGuuuGuuAuuAuGuAuGuAuuAuuGuGuAuGAuCuAGGuuAuGuuuuAuu
GuGuAuuuuAAuUGuuuAAuGuuGAuuuuuGAuuuuuuAuuAuuuuGuuuG*UUUGAuuu
GuAuuuGuuuGuuGGuuuGuG***UUUGuuuuuAuuGuuGuGGuuuAuGuuGuuuAAuuu
AuAuAGuuuAAUUUUGuAuuA*UUGuAuuACuUAUUUG***AAuuuG*UAuuUGuuGuuu
uGuAuuGuuuuuuuAuuGuAuAuuGCAuuuuuAuuuuuGuuuuGuuuuuuAuGuGAuuuu
uuuuuGuuuAAuAAuuuGuUAGuuGGuGAuA****GuuuuAuGGAuGuuuuuuuuAUUC*
*GuuuuuuGuuGuGuuuuuuAGAGuGuuuuuCuuuGuuGuGuCGuuGuuuGuCGACGuuu
uuGCGuuuGUUUUGuAAuuuAuuAuCAuCCCAuUUUUUAuuGuuGAuGuuuuuuGAuuuu
uuuUAuuuuAuuuuuGuuuuuuuuuuuuAuGGuGuuuuuuGuuAuuGAuuuAuuuuAuuu
AuuuuuGuGuuuuGuuuuuGuuuAuuAuuuuAUGuGuuuuuAuAuUUGuuGGAuuuAuUU
GCC***GCCAuAuuAC****AGuuAuuuAuuuuuuGuAAuAuGAuuuuGCAGuuGAuAAu
GG**AuuuuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGuAuuuGAuuuuuGAuAGuuAuu
AuAuuGuuGuuGAAAuuuG**GuuUGuuA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGU
UAAUAAUA

Courtesy of K. Stuart
 Structure of a Guide RNA (gRNA)

A6 Pre-mRNA
...AAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGUUGUGAUUUUGGAGUUAUAG...
|·||||||||||
UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
3’ 5’
U Tail Information Anchor
gA6[14] gRNA

...UUAGGGGGAGGAGAGAuAGuuAuuAuAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG...
····|··|·|·|||·||||·||||···|||··||||·||·||·|||||·||||||||||
UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
3’ 5’
U Tail Information Anchor

Courtesy of K. Stuart
 Two Models for Editing Directed by One gRNA

1. The Zipper
Editing Block
...UUAGGGGGAGGAGAGAuAGuuAuuAuAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG...
...AAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGUUGUGAUUUUGGAGUUAUAG...
...AAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGUUGUGuuAUUGGAGUUAUAG...
...AGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGUUGuuUGuuAUUGGAGUUAUAG...
...AAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGGuuUGuuAUUGGAGUUAUAG...
...GAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAuuuGGuuUGuuAUUGGAGUUAUAG...
...GCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG...
...AGGAAAGGUUAGGGGGAGGAGAGAAGAAAGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG...
...GAAAGGUUAGGGGGAGGAGAGAAGAAAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG...
...AAAGGUUAGGGGGAGGAGAGAAGAAuAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG...
...AGGUUAGGGGGAGGAGAGAAGAuuAuAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG...
...GUUAGGGGGAGGAGAGAAGuuAuuAuAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG...
...AAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGUUGUGAUUGGAGUUAUAG...
····|··|·|·|||·||||·||||···|||··||||·||·||·|||||·||||||||||
|·||||·||||···|||··||||·||·||·|||||·||||||||||
||·||||···|||··||||·||·||·|||||·||||||||||
||||···|||··||||·||·||·|||||·||||||||||
||···|||··||||·||·||·|||||·||||||||||
··|||··||||·||·||·|||||·||||||||||
||··||||·||·||·|||||·||||||||||
·||||·||·||·|||||·||||||||||
|·||·|||||·||||||||||
·||·|||||·||||||||||
·|||||·||||||||||
||·||||||||||
|·||||||||||
UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
UUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
UAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
UAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
UAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
UGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
CAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
UUUUAGACUAAGCAAUAGCCUCAAUAUC...
CUAAGCAAUAGCCUCAAUAUC...
UAAGCAAUAGCCUCAAUAUC...
GCAAUAGCCUCAAUAUC...
UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAA
UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAA
UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGAC
UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGA
UUUUUUUUUUUUAUUAAUAGUAUAGUGACAG
UUUUUUUUUUUUAUUAAUAGUAUAGUGA
UUUUUUUUUUUUAUUAAUAGUAUAG
UUUUUUUUUUUUAUUAAUAGUA
UUUUUUUUUUUUAUUAAUAG
UUUUUUUUUUUUAUUAA
UUUUUUUUUUUUA UAGCCUCAAUAUC...

U Tail Information Anchor

2. Dynamic Realignment

...UUAGGGGGAGGAGAGAuAGuuAuuAuAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG...
...AAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGUUGUGAUUUUGGAGUUAUAG...
...GCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGuuGAAA---GUUGUGAUUUUGGAGUUAUAG...
...AGGUUAGGGGGAGGAGAGAAGAuuAuA----GGuuGAAA---GUUGUGAUUUUGGAGUUAUAG...
...AGGUUAGGGGGAGGAGAGAAGAuuAuA----GGuuGAAA-GUUGuuUGAUUUUGGAGUUAUAG...
...AGGUUAGGGGGAGGAGAGAAGAuuAuAuuGuuGuuGAAA-GUUGuuUGAUUUUGGAGUUAUAG...
...UUAGGGGGAGGAGAGAuAGuuAuuAuAuuGuuGuuGAAA-GUUGuuUGAUUUUGGAGUUAUAG...
...UUAGGGGGAGGAGAGAuAGuuAuuAuAuuGuuGuuGAAA-GUUGuuUGuuAUUGGAGUUAUAG...
····|··|·|·|||·||||·||||···|||··|||
····|··|·|·|||·||||·||||···|||··||||·||·||·|||||·||||||||||
||·|||
||·||||···|||··|||
||··||| ·||·|
·||·|||||·||||||||||
|·||||||||||
UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUU
UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
UAGUAUAGUGACAGUUUUAGAC
UAGUAUAGUGACAGUUUU
UUUUUUUUUUUUAUUAAUAGUAUAGUGA
UUUUUUUUUUUUAUUAA UAAGCAAUAGCCUCAAUAUC...
UAAGCAAUAGCCUCAAUAUC...
UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAU
AGACUAAGCAAU

U Tail Information Anchor

Courtesy of K. Stuart
 Editing Proceeds in the 3' → 5' Direction

Editing of a downstream region is required for binding of

the subsequent gRNA’s anchor region.

Editing Domain
5' 5’ 5’ 5’ Block 3 Block 2 Block 1 3'

|||||||||||||||||||||
|||||||||||||| |||||||
|||||||||||||| |||||||
UUUUU UUUUU UUUUU

Courtesy of K. Stuart
 edited T. brucei ATPase subunit 6 RNA
edited
editing in progress...
pre-edited

M F L F F F C D
AAAAAUAAGUAUUUUGAUAUUAUUAAAGUAAAAGAGGAAUUUUGGGCGGAAGAGAAGGA
AAAAAUAAGUAUUUUGAUAUUAUUAAAGUAAAAGuuuuuAuuuuuuuuuuGuGAuuuAU
AAAAAUAAGUAUUUUGAUAUUAUUAAAGUAAAuAuGuuuuuAuuuuuuuuuuGuGAuuu
L F W L R L M LK LE CK MG YF Y E C R V G W V S F RW LG C E FE
GACAGGAGAGGAAAUGAAGGAGAAAGGUUUUGAGAGGGGGGUUUUUUGAGGGGAGGAAA
GACAGGAGAGGAAAUGAAGGAGAAAGGUUUUGAGAGGGGGGUUUUUUGAGuuGuGGuuu
GACAGGAGAGGAuuuuAAuUGuuuAAuGuuGAuuuuuGAuuuuuuAuuAuuuuGuuuG*
UUUGGuuGCGuuuGuuAuuAuGuAuGuAuuAuuGuGuAuGAuCuAGGuuAuGuuuuAuu
AUUUUGGuuGCGuuuGuuAuuAuGuAuGuAuuAuuGuGuAuGAuCuAGGuuAuGuuuuA
IK VE YF FW NI CW LT MI LC IL FS D Y F G L R L E F A CR LR RF
AAGAAUUUUGAAUUUGAACUAUUUGUUUAAGUUAUGGGAGAGAAGCAAGGAGGAGAAAA
AAGAAUUUUGAAUUUGAACUAUUUGUUUAAGUUAUGGGAGAGAuAuuGCAuuuuuAuuu
AAGAAUUUUGAAUUUGAACuUAUUUG***AAuuuG*UAuuUGuuGuuuuGuAuuGuuuu
AuGuuGuuuAAuuuAuAuAGuuuAAUUUUGuAuuA*UUGuAuuACuUAUUUG***AAuu
UUUGAuuuGuAuuuGuuuGuuGGuuuGuG***UUUGuuuuuAuuGuuGuGGuuuAuGuu
GuGuAuuuuAAuUGuuuAAuGuuGAuuuuuGAuuuuuuAuuAuuuuGuuuG*UUUGAuu
uuGuGuAuuuuAAuUGuuuAAuGuuGAuuuuuGAuuuuuuAuuAuuuuGuuuG*UUUGA
KD VL GY EL FF WV GG DL SC W GL EF AL GL GL RW RF RM FL
GUAGGGGAAUUUUGAGGAGAUUCUUGGGGAGAGGCGGGCGGGCGACGGCGGUUUUGAAA
GUAGGGGAAUUUUGAGGAGuuuuuuuuAUUC**GuuuuuuGuuGuGuuuuuuAGAGuGu
uuGuuuuGuuuuuuAuGuGAuuuuuuuuuGuuuAAuAAuuuGuUAGuuGGuGAuA****
uuuAuuGuAuAuuGCAuuuuuAuuuuuGuuuuGuuuuuuAuGuGAuuuuuuuuuGuuuA
uG*UAuuUGuuGuuuuGuAuuGuuuuuuuAuuGuAuAuuGCAuuuuuAuuuuuGuuuuG
GuuuAAuuuAuAuAGuuuAAUUUUGuAuuA*UUGuAuuACuUAUUUG***AAuuuG*UA
uGuAuuuGuuuGuuGGuuuGuG***UUUGuuuuuAuuGuuGuGGuuuAuGuuGuuuAAu
uuuGuAuuuGuuuGuuGGuuuGuG***UUUGuuuuuAuuGuuGuGGuuuAuGuuGuuuA
WF KN HL PY FS LL GI GL ter
Y EY G C R I K T G Y E L M E N L L G YI
ACACCCAUUUUUAGGAGGAUAAGAGGGGAGAAAAGGGGAAAUGGAAUUGGGAAUUGCCU
ACACCCAUUUUUAGGAGGAUAAGAGGGGAGAAAAGGGGAAAUGGAAuUUGuuGGAuuuA
ACACCCAUUUUUAGGAGGAUAAGAGGGGAGAAAAGGGGuuuAuuAuuuuAUGuGuuuuu
ACACCCAUUUUUAGGAGGAUAAGAGGGuuuuuuGuuAuuGAuuuAuuuuAuuuAuuuuu
ACACCCAUUUUUAGGAGGAUAuuuuAuuuuuGuuuuuuuuuuuuAGGGuuuuuuGuuAu
uuuuCuuuGuuGuGuCGuuGuuuGuCGACGuuuuuGCGuuuGUUUUGuAAuuuAuuAuC
GuuuuAuGGAuGuuuuuuuuAUUC**GuuuuuuGuuGuGuuuuuuAGAGuGuuuuuCuu
AuAAuuuGuUAGuuGGuGAuA****GuuuuAuGGAuGuuuuuuuuAUUC**GuuuuuuG
uuuuuuAuGuGAuuuuuuuuuGuuuAAuAAuuuGuUAGuuGGuGAuA****GuuuuAuG
uuUGuuGuuuuGuAuuGuuuuuuuAuuGuAuAuuGCAuuuuuAuuuuuGuuuuGuuuuu
uuAuAuAGuuuAAUUUUGuAuuA*UUGuAuuACuUAUUUG***AAuuuG*UAuuUGuuG
AuuuAuAuAGuuuAAUUUUGuAuuA*UUGuAuuACuUAUUUG***AAuuuG*UAuuUGu
LA LF FA CK IL VL FE LE LR YA IG A K F V L R F G L R FR CE F E
UUGCCAAACUUUUAGAAGAAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAA
UUGCCAAACUUUUAGAAGAAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGuuGu
UUGCCAAACUUUUAGAAGAAAGAGCAGGAAAGGUUAGGGGGuuuAGuuuuGuAuuuGAu
UUGCCAAACUUUUAGAAGAAAGAGCAGGAAAGG**AuuuuuuGuuGuuuuuGuuGuuuG
UUuGCC***GCCAuAuuAC****AGuuAuuuAuuuuuuGuAAuAuGAuuuuGCAGuuGA
AuAuUUGuuGGAuuuAUUuGCC***GCCAuAuuAC****AGuuAuuuAuuuuuuGuAAu
GuGuuuuGuuuuuGuuuAuuAuuuuAUGuGuuuuuAuAuUUGuuGGAuuuAUUuGCC**
uGAuuuAuuuuAuuuAuuuuuGuGuuuuGuuuuuGuuuAuuAuuuuAUGuGuuuuuAuA
AuCCCAuUUUUUAuuGuuGAuGuuuuuuGAuuuuuuuUAuuuuAuuuuuGuuuuuuuuu
uGuuGuGuCGuuGuuuGuCGACGuuuuuGCGuuuGUUUUGuAAuuuAuuAuCAuCCCAu
uuGuGuuuuuuAGAGuGuuuuuCuuuGuuGuGuCGuuGuuuGuCGACGuuuuuGCGuuu
GAuGuuuuuuuuAUUC**GuuuuuuGuuGuGuuuuuuAGAGuGuuuuuCuuuGuuGuGu
uAuGuGAuuuuuuuuuGuuuAAuAAuuuGuUAGuuGGuGAuA****GuuuuAuGGAuGu
uuuuGuAuuGuuuuuuuAuuGuAuAuuGCAuuuuuAuuuuuGuuuuGuuuuuuAuGuGA
uGuuuuGuAuuGuuuuuuuAuuGuAuAuuGCAuuuuuAuuuuuGuuuuGuuuuuuAuGu
L
R C E DS FC FD LF FG NV NI LE ter
L V D G Q D I S terter
S F M D
AGUUGUGAUUUUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAUAAUA
AGUUGUGA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAUAAUA
AGUUGuuUGuuA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAUAAUA
uGAAAuuuG**GuuUGuuA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAUAAUA
uuuuGAuAGuuAuuAuAuuGuuGuuGAAAuuuG**GuuUGuuA**UUGGAGUUAUAGAA
uuuAGuuuuGuAuuuGAuuuuuGAuAGuuAuuAuAuuGuuGuuGAAAuuuG**GuuUGu
uAAuGG**AuuuuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGuAuuuGAuuuuuGAuAG
AuGAuuuuGCAGuuGAuAAuGG**AuuuuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGu
*GCCAuAuuAC****AGuuAuuuAuuuuuuGuAAuAuGAuuuuGCAGuuGAuAAuGG**
uUUGuuGGAuuuAUUuGCC***GCCAuAuuAC****AGuuAuuuAuuuuuuGuAAuAuG
uuuAGGGuuuuuuGuuAuuGAuuuAuuuuAuuuAuuuuuGuGuuuuGuuuuuGuuuAuu
UUUUUAuuGuuGAuGuuuuuuGAuuuuuuuUAuuuuAuuuuuGuuuuuuuuuuuuAGGG
GUUUUGuAAuuuAuuAuCAuCCCAuUUUUUAuuGuuGAuGuuuuuuGAuuuuuuuUAuu
CGuuGuuuGuCGACGuuuuuGCGuuuGUUUUGuAAuuuAuuAuCAuCCCAuUUUUUAuu
uuuuuuuAUUC**GuuuuuuGuuGuGuuuuuuAGAGuGuuuuuCuuuGuuGuGuCGuuG
uuuuuuuuuGuuuAAuAAuuuGuUAGuuGGuGAuA****GuuuuAuGGAuGuuuuuuuu
GAuuuuuuuuuGuuuAAuAAuuuGuUAGuuGGuGAuA****GuuuuAuGGAuGuuuuuu
V F F I R F L L C F L E C F S L L C R
UAAGAUCAAAUAAGUUAAUAAUA
uA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAUAAUA
uuAuuAuAuuGuuGuuGAAAuuuG**GuuUGuuA**UUGGAGUUAUAGAAUAAGAUCAA
AuuuGAuuuuuGAuAGuuAuuAuAuuGuuGuuGAAAuuuG**GuuUGuuA**UUGGAGU
AuuuuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGuAuuuGAuuuuuGAuAGuuAuuAuA
AuuuuGCAGuuGAuAAuGG**AuuuuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGuAuu
AuuuuAUGuGuuuuuAuAuUUGuuGGAuuuAUUuGCC***GCCAuAuuAC****AGuuA
uuuuuuGuuAuuGAuuuAuuuuAuuuAuuuuuGuGuuuuGuuuuuGuuuAuuAuuuuAU
uuAuuuuuGuuuuuuuuuuuuAGGGuuuuuuGuuAuuGAuuuAuuuuAuuuAuuuuuGu
GuuGAuGuuuuuuGAuuuuuuuUAuuuuAuuuuuGuuuuuuuuuuuuAGGGuuuuuuGu
uuuGuCGACGuuuuuGCGuuuGUUUUGuAAuuuAuuAuCAuCCCAuUUUUUAuuGuuGA
AUUC**GuuuuuuGuuGuGuuuuuuAGAGuGuuuuuCuuuGuuGuGuCGuuGuuuGuCG
uuAUUC**GuuuuuuGuuGuGuuuuuuAGAGuGuuuuuCuuuGuuGuGuCGuuGuuuGu
C L S T F L R L F C N L L S S H F L L
AUAAGUUAAUAAUA
UAUAGAAUAAGAUCAAAUAAGUUAAUAAUA
uuGuuGuuGAAAuuuG**GuuUGuuA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUA
uGAuuuuuGAuAGuuAuuAuAuuGuuGuuGAAAuuuG**GuuUGuuA**UUGGAGUUAU
uuuAuuuuuuGuAAuAuGAuuuuGCAGuuGAuAAuGG**AuuuuuuGuuGuuuuuGuuG
GuGuuuuuAuAuUUGuuGGAuuuAUUuGCC***GCCAuAuuAC****AGuuAuuuAuuu
GuuuuGuuuuuGuuuAuuAuuuuAUGuGuuuuuAuAuUUGuuGGAuuuAUUuGCC***G
uAuuGAuuuAuuuuAuuuAuuuuuGuGuuuuGuuuuuGuuuAuuAuuuuAUGuGuuuuu
uGuuuuuuGAuuuuuuuUAuuuuAuuuuuGuuuuuuuuuuuuAGGGuuuuuuGuuAuuG
ACGuuuuuGCGuuuGUUUUGuAAuuuAuuAuCAuCCCAuUUUUUAuuGuuGAuGuuuuu
CGACGuuuuuGCGuuuGUUUUGuAAuuuAuuAuCAuCCCAuUUUUUAuuGuuGAuGuuu
L M F F D F F Y F I F V F F F W C F L
AUAAUA
AGAAUAAGAUCAAAUAAGUUAAUAAUA
uuuGuuuAGuuuuGuAuuuGAuuuuuGAuAGuuAuuAuAuuGuuGuuGAAAuuuG**Gu
uuuGuAAuAuGAuuuuGCAGuuGAuAAuGG**AuuuuuuGuuGuuuuuGuuGuuuGuuu
CCAuAuuAC****AGuuAuuuAuuuuuuGuAAuAuGAuuuuGCAGuuGAuAAuGG**Au
AuAuUUGuuGGAuuuAUUuGCC***GCCAuAuuAC****AGuuAuuuAuuuuuuGuAAu
AuuuAuuuuAuuuAuuuuuGuGuuuuGuuuuuGuuuAuuAuuuuAUGuGuuuuuAuAuU
uGAuuuuuuuUAuuuuAuuuuuGuuuuuuuuuuuuAGGGuuuuuuGuuAuuGAuuuAuu
uuuGAuuuuuuuUAuuuuAuuuuuGuuuuuuuuuuuuAGGGuuuuuuGuuAuuGAuuuA
L L I Y F I Y F C V L F L F I I L C V F
uUGuuA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAUAAUA
AGuuuuGuAuuuGAuuuuuGAuAGuuAuuAuAuuGuuGuuGAAAuuuG**GuuUGuuA*
uuuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGuAuuuGAuuuuuGAuAGuuAuuAuAuu
AuGAuuuuGCAGuuGAuAAuGG**AuuuuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGu
UGuuGGAuuuAUUuGCC***GCCAuAuuAC****AGuuAuuuAuuuuuuGuAAuAuGAu
uuAuuuAuuuuuGuGuuuuGuuuuuGuuuAuuAuuuuAUGuGuuuuuAuAuUUGuuGGA
uuuuAuuuAuuuuuGuGuuuuGuuuuuGuuuAuuAuuuuAUGuGuuuuuAuAuUUGuuG
I F V G F I C R H I T V I Y F L ter
*UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAUAAUA
GuuGuuGAAAuuuG**GuuUGuuA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAU
AuuuGAuuuuuGAuAGuuAuuAuAuuGuuGuuGAAAuuuG**GuuUGuuA**UUGGAGU
uuuGCAGuuGAuAAuGG**AuuuuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGuAuuuG
uuuAUUuGCC***GCCAuAuuAC****AGuuAuuuAuuuuuuGuAAuAuGAuuuuGCAG
GAuuuAUUuGCC***GCCAuAuuAC****AGuuAuuuAuuuuuuGuAAuAuGAuuuuGC
AAUA
UAUAGAAUAAGAUCAAAUAAGUUAAUAAUA
AuuuuuGAuAGuuAuuAuAuuGuuGuuGAAAuuuG**GuuUGuuA**UUGGAGUUAUAG
uuGAuAAuGG**AuuuuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGuAuuuGAuuuuuG
AGuuGAuAAuGG**AuuuuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGuAuuuGAuuuu
AAUAAGAUCAAAUAAGUUAAUAAUA Bhat et al. (1990)
uGAuAGuuAuuAuAuuGuuGuuGAAAuuuG**GuuUGuuA**UUGGAGUUAUAGAAUAA
AuAGuuAuuAuAuuGuuGuuGAAAuuuG**GuuUGuuA**UUGGAGUUAUAGAAUAAGA
UCAAAUAAGUUAAUAAUA
GAUCAAAUAAGUUAAUAAUA
 RNA editing in kinetoplastid mitochondria
gRNA 1

~1kb
gRNA 2

gRNA 3
ND8
ND7 MINICIRCLE
COIII (>1000 different molecules)
ND9 CYB
A6

22kb CR3 1000s of gRNAs

Courtesy of K. Stuart

CR5 COII
CR4
MURF2 U
RPS12

MAXICIRCLE
U
(~50 copies)

U
12 pre-edited RNAs
 Pre-mRNA - nonsense ORF
Corresponds to genome sequence
Contains only one gRNA anchor site

Anchor Site 1
Edits 1..?
U

U
Numerous
distinct
Site 2, Edits gRNAs
Intermediate mRNAs,
partially correspond to U
Site 3, Edits
genome, contain some
gRNA anchor sites U

Mature mRNA - sensible ORF

Does not correspond to genome sequence
Contains all gRNA anchor sites
 Issue #4
• Do we need to create RNA
intermediates?
– Currently, we don’t have mRNA splice
intermediates
– A gRNA only has an anchor site on an
edited RNA (except first gRNA), It cannot
be mapped to the Maxi-circle DNA directly
• Do we need to label/capture the 3'→5’
direction of processing? Do other
systems do it differently?
 Guide_RNA

Primary_protein_coding_trancript

intermediate_edited_mRNA Insert_U
part_of
intersection_of: mRNA
intersection of: associated_with
guide_RNA Intermediate_edited_mRNA
intersection_of: part_of edit
Intersection_of part_of
anchored_region delete_U
intersection_of has_quality edited
Intermediate_edited_mRNA
guide_RNA
part_of anchor
delete_U delete_U part_of
insert_U is_a edit_operation Insert_U Insert_U
delete_U is_a edit_operation
edited_mRNA
edited_mRNA
intersection of:mRNA
intersection of: has_quality edited dervies_from???
intersection_of part_of edit
edited_CDS
edited_CDS derives_from
edited_mRNA
dervies_from
edited_polypeptide derives_from
edited_CDS
edited_polypeptide